peerj_json_files/PeerJ_Json_130.json

{
    "v1_Abstract": "How avifauna respond to the long-term loss and fragmentation of tropical forests is a critical issue in biodiversity management. We use data from over 30 years to gain insights into such changes in the northernmost Neotropical rainforest in the Sierra de Los Tuxtlas of southern Veracruz, Mexico. This region has been extensively deforested over the past half-century. The Estaci\u00f3n de Biolog\u00eda Tropical Los Tuxtlas, of the Universidad Nacional Aut\u00f3noma de M\u00e9xico (UNAM), protects a 640 ha tract of lowland forest. It became relatively isolated from other forested tracts between 1975 and 1985, but it retains a corridor of forest to more extensive forests at higher elevations on Volc\u00e1n San Mart\u00edn. Most deforestation in this area occurred during the 1970s and early 1980s. Forest birds were sampled on the station and surrounding areas using mist nets during eight non-breeding seasons from 1973 to 2004 (though in some seasons netting extended into the local breeding season for some species). Our data suggested extirpations or declines in 12 species of birds subject to capture in mist nets. Six of the eight species no longer present were captured in 1992-95, but not in 20032004. Presence/absence data from netting and observational data suggested that another four low-density species also disappeared since sampling began. This indicates a substantial time lag between the loss of habitat and the apparent extirpation of these species. Delayed species loss and the heterogeneous nature of the species affected will be important factors in tropical forest management and conservation.",
    "v1_col_introduction": "introduction  : Deforestation is one of the main threats to biodiversity conservation. Forest loss\nand fragmentation have caused declines or local extinctions among animal species at many locations (Turner, 1996; Fahrig, 2003; Dirzo & Raven, 2003). Local population declines and extiprations may be the most important leading indicators of biodiversity loss (Ceballos & Ehrlich, 2002; O\u2019Grady et al., 2004). Bird losses have been documented in many forest systems (e.g., Willis, 1974, 1979; Leck, 1979; Karr, 1982; Bierregaard & Lovejoy, 1989; Kattan, Halvarez-Lopez, & Giraldo, 1994; Robinson, 1999; Sodhi, Liow, & Bazzaz, 2004; Ferraz et al., 2007; Patten, G\u00f3mez de Silva, & Smith-Patten, 2010; Laurance et al., 2011). Perhaps nowhere has this phenomenon been more noticeable than among tropical forests, where species losses have been documented in numerous taxonomic groups (e.g., Zimmerman & Bierregaard, 1986; Powell & Powell, 1987; Malcolm, 1988; Pahl, Winter, & Heinsohn 1988; Becker, Moure, & Peralta, 1991; Daily & Ehrlich, 1995; Brook, Sodhi, & Ng, 2003, Dirzo & Raven, 2003, Stuart et al. 2004; Robinson & Sherry, 2012). Species losses can occur at the landscape or patch levels and depend on the intensity of the change in forest cover, the distance to and size of other forest fragments, shape and size of the fragment, and other factors (Robbins, 1980; Lovejoy et al., 1984, 1986, Rolstad, 1991; Andr\u00e9n, 1994; Faaborg et al., 1995; Lees & Peres, 2006; Barlow et al., 2006; Patten & Smith-Patten, 2011; Robinson & Sherry 2012). Tropical forest species, which often occur in small, low-density populations, may be particularly vulnerable to extirpation (Terborgh & Winter 1980; Pimm, Jones, & Diamond, 1988; Stotz et al., 1996).\nRelatively few studies have assessed changes through decades, however (Ewers &\nDidham, 2006). And although deforestation and fragmentation can occur over a short period, some time may pass before species begin to disappear from an affected area\n28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52\nPeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013)\nR ev ie w in g M an\nus cr ip t\n(Leigh, 1975, 1981; Karr, 1982, Tilman et al., 1994; Brooks, Pimm, & Oyugi, 1999). Thus, to fully document the impact of deforestation on a forest community, a site must be studied for a substantial period of time after habitat alteration has occurred. Detailing the process of local population decline and extirpation over time provides invaluable information about species\u2019 abilities to cope with habitat fragmentation. It also informs us about how community composition itself may be resistant to change, its degree of resilience following change, and how or if it stabilizes following this disturbance.\nStudies of species losses in birds have used a variety of methods, including\ncomparing species richness in different-sized fragments (Willis, 1979; Nemark, 1991; Blake, 1991), comparison of species composition at a site pre- and post-fragmentation (Willis, 1974; Leck, 1979; Bierregaard & Lovejoy, 1989; Kattan, 1994; Patten & SmithPatten, 2011), and experimental fragmentation (Lovejoy et al., 1986; Bierregaard & Lovejoy, 1988, 1989; Ferraz et al., 2003, 2007; Laurance et al., 2011), and have often included scattered survey data prior to fragmentation (Willis, 1974; Leck, 1979; Kattan, Halvarez-Lopez, & Giraldo, 1994; Robinson, 1999; Patten, G\u00f3mez de Silva, & SmithPatten, 2010; Patten & Smith-Patten, 2011). Many of these studies have relied on qualitative visual and audio survey techniques, with multiple observers, though such techniques can allow cryptic and low-density species to be overlooked (Whitman, Hagan, & Brokaw, 1997). Additionally, observer skills and intensity of sampling may vary among surveys.\nMist netting offers the most consistent and quantitative method available to\nsample birds among years (Rappole, Winker, & Powell, 1998). However, mist nets have documented weaknesses; the most relevant is the limited stratum and size of birds they can effectively sample (Remsen & Good, 1996; Whitman, Hagan, & Brokaw, 1997; Rappole, Winker, & Powell, 1998). This is particularly noticeable in structurally diverse\n53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77\nPeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013)\nR ev ie w in g M an\nus cr ip t\nhabitats such as tropical rainforests, where probability of detection using mist nets is unknown for most species. Mist net studies in the Neotropics are therefore biased toward understory, small- to mid-sized passerines. While mist nets, unlike other methods, are less prone to observer bias and variability, we augmented our analyses of netting data that suggested species losses with presence-absence observational data (daily checklists in later years); this becomes particularly important for low-density species and for those not readily captured.\nThe Sierra de Los Tuxtlas of southern Veracruz, Mexico provides a textbook case\nof deforestation. This small range of volcanic mountains is home to the northernmost Neotropical rainforest (Pennington & Sarukhan, 1968; Dirzo & Miranda, 1991). The region has lost more than 90% of its forests in the past century, with the majority of that loss occurring in the lowlands over the past fifty years (Dirzo & Garcia, 1992; Rappole, Powell, and Sader, 1994; Winker, 1997). Our study compares eight seasons of mist net sampling from Los Tuxtlas over the course of more than thirty years. This allows us to at least partly answer the question of how species composition and relative abundance changed in and around a conserved core of local rainforest habitat on a decadal scale. METHODS\nThe Sierra de Los Tuxtlas is located in southern Veracruz, Mexico, 90 km\nsoutheast of Veracruz city. This range of mountains lies in the northwestern portion of the Isthmus of Tehuantepec and is isolated from the Sierra Madre Oriental by extensive lowlands. Los Tuxtlas encompass approximately 4,200 km2, and the range is dominated by Volc\u00e1n San Mart\u00edn and Volc\u00e1n Santa Marta, each reaching more than 1,500 m elevation. The Gulf of Mexico lies a short distance from the mountains to the north and east. The northernmost Neotropical evergreen rainforest formerly dominated the habitat in the region (Andrle, 1966; Pennington & Sarukhan, 1968; Dirzo & Miranda, 1991), but\n78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102\nPeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013)\nR ev ie w in g M an\nus cr ip t\ndue to deforestation it is now a mosaic with a high percentage of pasture, cropland, fencerows, and isolated trees (pers. obs.; Dirzo & Garcia 1992; Estrada, Coates-Estrada, & Merritt 1997). Andrle (1966) estimated that 50% of the region was forested in 1962. By 1975 Rappole & Warner (1980) estimated that a third of the forests still stood. Just 15% of forest remained in 1986 (Winker, Rappole, & Ramos, 1990; Dirzo & Garcia, 1992), and in 1994 only 7-10% of the region was forested (Winker, 1997). Remaining forest occurs primarily in the highlands, and below 500 m forest is scarce (Rappole, Powell, & Sader, 1994; Mendoza, Fay, & Dirzo, 2005; Figs. 1, 2, S1).\nThe climate in Los Tuxtlas is warm and wet, with a mean annual temperature of\n25 C, and annual precipitation is 4,500-4,900 mm, with a short dry season from MarchMay (Soto & Gama 1997). Canopy heights in primary forest range from 30-35 m (IbarraManriquez et al., 1997). Second growth areas generally have variable canopy heights from 3-20 m (pers. obs.).\nIn 1967 the Universidad Nacional Aut\u00f3noma de M\u00e9xico established the Estaci\u00f3n\nde Biolog\u00eda Los Tuxtlas, protecting a 640-ha tract of lowland rainforest (Gonz\u00e1lezSoriano, Dirzo, & Vogt, 1997). Over the following decades this site became largely isolated from other tracts of forest, although a corridor of forest remains, connecting to the more extensive upland forests on Volc\u00e1n San Mart\u00edn (Dirzo & Garcia, 1992; Fig. 2). The first intensive sampling of birds in the region began in 1973, and data from that effort are included here (see Winker, 1997).\nDuring the non-breeding seasons of 1973-74 and 1974-75 Oehlenschlager, Ramos,\nRappole, and Warner conducted the first intensive mist-netting efforts in the area. Sites extended through what was then contiguous rainforest from the biological station eastward to the coast (Fig. 3). In 1986, Rappole, Ramos, and Winker operated mist nets at the biological station, and Winker and Escalante continued work there from 1992 to\n103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127\nPeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013)\nR ev ie w in g M an\nus cr ip t\n1994. In 2003-04 as part of a study of migrant birds, Shaw operated mist nets at the same location as Winker and Escalante\u2019s work in the 1990s. This study was approved by the University of Alaska Fairbanks IACUC (approval numbers: #00-33 & #04-03). Fieldwork occurred primarily during the non-breeding season. Effort was made to equally sample the available forest types throughout the study period, although, in order to do this, habitat changes precluded using the same sites across all years (see Winker 1995; Fig. 3). Field effort as gauged by net hours also varied among years (Table 1).\nOur earliest sampling occurred over a wider area than later seasons (Fig. 3).\nDuring the earliest sampling, large tracts of contiguous forest consisting of various microhabitats dominated the region and were sampled accordingly (Fig. 3). This broader expanse of forest likely provided habitat to more species than the current distribution of forest. This increased detection probabilities for some species such as Schiffornis turdina, which was rare even during our earliest sampling. Two general types of forest were present after fragmentation: primary forest and acahual (second growth). Because our sampling was forest-oriented, our efforts tracked the distribution of these habitats. Primary rainforest and second growth habitats were sampled in all efforts. We were unable to separate capture data by site for the early sampling periods; our findings therefore include data from the somewhat larger area from the station east to the coast. Our sampling was also uneven with respect to season, with wet and dry season sampling being unevenly distributed among years; we attempt to account for this, especially in relation to seasonal movements, when considering the results. This sampling heterogeneity leads us to be cautious and conservative in our analyses and\ninterpretations. Importantly, however, the same site (18\u00b0 34\u201950\u201dN, 95\u00b0 04\u201920\u201dW) and net\nlanes were used in the 1992-2004 efforts (sample periods 4-8 in Table 1).\nOnly resident species were used in our analyses due to seasonal migration and the\n128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149\n150 151 152\nPeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013)\nR ev ie w in g M an\nus cr ip t\nhigh levels of variance in abundance this causes among obligate migrants. Changes in relative abundance were detected by comparing capture rates (birds per 1000 net hours) from each year of sampling. Through visual inspection of data (Appendix) we chose species absent in later samples and those with trends of apparently declining or increasing rates of capture for more detailed analyses. Neither gaps nor monotonic changes were necessary for inclusion, just suggestion of a possible trend. We did this instead of applying statistical tests across all 122 species to minimize Type I and Type II errors either by applying a very large number of tests or a conservative correction (e.g., Bonferroni). Presence/absence patterns and observational data (daily checklists in later years) were also considered to provide insight into changes in abundance in low-density species that did not have sufficient samples for statistical testing. Species were considered for examination for presence/absence if they had not been captured since at least 1986-87. Vagrants, defined as those rarely encountered species whose ranges do not normally include the Sierra de Los Tuxtlas, were excluded (Winker et al., 1992; Howell & Webb, 1995). Only first-time captures (within a season) were used in statistical analyses. Ordinary least squares regression was used to detect changes in abundance for selected species. We looked for newly appearing species using presence/absence netting, observational, and specimen data. Daily checklists were used to augment mist-net data as a check to determine whether absence from the mist-net data was indicative of reality.\nSpecies showing statistically significant declines and those not captured or\nobserved in later sampling periods were categorized by preferred habitat (edge, forest, or semi-open), food preference (fruit/nectar or insects), elevational range, and whether Los Tuxtlas was at the periphery or core of its geographic range (Howell & Webb, 1995). These characteristics were used to assess whether certain traits of the species increased their vulnerability to local extirpation.\n153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177\nPeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013)\nR ev ie w in g M an\nus cr ip t",
    "v2_Abstract": "How avifauna respond to the long-term loss and fragmentation of tropical forests is a critical issue in biodiversity management. We use data from over 30 years to gain insights into such changes in the northernmost Neotropical rainforest in the Sierra de Los Tuxtlas of southern Veracruz, Mexico. This region has been extensively deforested over the past half-century. The Estaci\u00f3n de Biolog\u00eda Tropical Los Tuxtlas, of the Universidad Nacional Aut\u00f3noma de M\u00e9xico (UNAM), protects a 640 ha tract of lowland forest. It became relatively isolated from other forested tracts between 1975 and 1985, but it retains a corridor of forest to more extensive forests at higher elevations on Volc\u00e1n San Mart\u00edn. Most deforestation in this area occurred during the 1970s and early 1980s. Forest birds were sampled on the station and surrounding areas using mist nets during eight non-breeding seasons from 1973 to 2004 (though in some seasons netting extended into the local breeding season for some species). Our data suggested local extinctions or declines in 12 species of birds subject to capture in mist nets. Six of the eight species no longer present were captured in 1992-95, but not in 2003-2004. Presence/absence data from netting and observational data suggested that another four low-density species also disappeared since sampling began. This indicates a substantial time lag between the loss of habitat and the apparent extirpation of these species. Delayed species loss and the heterogeneous nature of the species affected will be important factors in tropical forest management and conservation.",
    "v2_col_introduction": "introduction  : Deforestation is one of the main threats to biodiversity conservation.\nForest loss and fragmentation have caused declines or local extinctions among animal species at many locations (Turner, 1996; Fahrig, 2003; Dirzo & Raven, 2003). Local population declines and extinctions may be the most important leading indicators of biodiversity loss (Ceballos & Ehrlich, 2002; O\u2019Grady et al., 2004). Bird losses have been documented in many forest systems (e.g., Willis, 1974, 1979; Leck, 1979; Karr, 1982; Bierregaard & Lovejoy, 1989; Kattan, Halvarez-Lopez, & Giraldo, 1994; Robinson, 1999; Sodhi, Liow, & Bazzaz, 2004; Ferraz et al., 2007; Patten, G\u00f3mez de Silva, & Smith-Patten, 2010; Laurance et al., 2011). Perhaps nowhere has this phenomenon been more noticeable than among tropical forests, where species losses have been documented in numerous taxonomic groups (e.g., Zimmerman & Bierregaard, 1986; Powell & Powell, 1987; Malcolm, 1988; Pahl, Winter, & Heinsohn 1988; Becker, Moure, & Peralta, 1991; Daily & Ehrlich, 1995; Brook, Sodhi, & Ng, 2003, Dirzo & Raven, 2003, Stuart et al. 2004; Robinson & Sherry, 2012). Species losses can occur at the landscape or patch levels and depend on the intensity of the change in forest cover, the distance to and size of other forest fragments, shape and size of the fragment, and other factors (Robbins, 1980; Lovejoy et al., 1984, 1986, Rolstad, 1991; Andr\u00e9n, 1994; Faaborg et al., 1995; Lees & Peres, 2006; Barlow et al., 2006; Robinson & Sherry 2012). Tropical forest species, which often occur in small, low-density populations, may be particularly vulnerable to local extinctions (Terborgh & Winter 1980; Pimm, Jones, & Diamond, 1988;\n30\n31\n32\n33\n34\n35\n36\n37\n38\n39\n40\n41\n42\n43\n44\n45\n46\n47\n48\n49\n50\n51\n52\n53\nPeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013)\nR ev ie w in g M an\nus cr ip t\nStotz et al., 1996).\nRelatively few studies have assessed changes through decades,\nhowever (Ewers & Didham, 2006). And although deforestation and fragmentation can occur over a short period, some time may pass before species begin to disappear from an affected area (Leigh, 1975, 1981; Karr, 1982, Tilman et al., 1994; Brooks, Pimm, & Oyugi, 1999). Thus, to fully document the impact of deforestation on a forest community, a site must be studied for a substantial period of time after habitat alteration has occurred. Detailing the process of local population decline and extinction over time provides invaluable information about species\u2019 abilities to cope with habitat fragmentation and how community composition itself stabilizes following this disturbance.\nStudies of species losses in birds have used a variety of methods,\nincluding comparing species richness in different-sized fragments (Willis, 1979; Nemark, 1991; Blake, 1991), comparison of species composition at a site pre- and post-fragmentation (Willis, 1974; Leck, 1979; Bierregaard & Lovejoy, 1989; Kattan, 1994), and experimental fragmentation (Lovejoy et al., 1986; Bierregaard & Lovejoy, 1988, 1989; Ferraz et al., 2003, 2007; Laurance et al., 2011), and have often included scattered survey data prior to fragmentation (Willis, 1974; Leck, 1979; Kattan, Halvarez-Lopez, & Giraldo, 1994; Robinson, 1999; Patten, G\u00f3mez de Silva, & Smith-Patten, 2010). Many of these studies have relied on qualitative visual and audio survey techniques, with multiple observers, though such techniques can allow cryptic and low-density species to be overlooked (Whitman, Hagan, &\n54\n55\n56\n57\n58\n59\n60\n61\n62\n63\n64\n65\n66\n67\n68\n69\n70\n71\n72\n73\n74\n75\n76\n77\nPeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013)\nR ev ie w in g M an\nus cr ip t\nBrokaw, 1997). Additionally, observer skills and intensity of sampling may vary among surveys.\nMist netting offers the most consistent and quantitative method\navailable to sample birds among years (Rappole, Winker, & Powell, 1998). However, mist nets have documented weaknesses; the most relevant is the limited stratum and size of birds they can effectively sample (Remsen & Good, 1996; Whitman, Hagan, & Brokaw, 1997; Rappole, Winker, & Powell, 1998). This is particularly noticeable in structurally diverse habitats such as tropical rainforests, where probability of detection using mist nets is unknown for most species. Mist net studies in the Neotropics are therefore biased toward understory, small- to mid-sized passerines. While mist nets, unlike other methods, are less prone to observer bias and variability, we augmented our analyses of netting data that suggested species losses with presence-absence observational data (daily checklists in later years); this becomes particularly important for low-density species and for those not readily captured.\nThe Sierra de Los Tuxtlas of southern Veracruz, Mexico provides a\ntextbook case of deforestation. This small range of volcanic mountains is home to the northernmost Neotropical rainforest (Pennington & Sarukhan, 1968; Dirzo & Miranda, 1991). The region has lost more than 90% of its forests in the past century, with the majority of that loss occurring in the lowlands over the past fifty years (Dirzo & Garcia, 1992; Rappole, Powell, and Sader, 1994; Winker, 1997). Our study compares eight seasons of mist net sampling from Los Tuxtlas over the course of more than thirty years. This\n78\n79\n80\n81\n82\n83\n84\n85\n86\n87\n88\n89\n90\n91\n92\n93\n94\n95\n96\n97\n98\n99\n100\n101\nPeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013)\nR ev ie w in g M an\nus cr ip t\nallows us to at least partly answer the question of how species composition and relative abundance changed in and around a conserved core of local rainforest habitat on a decadal scale. METHODS\nThe Sierra de Los Tuxtlas is located in southern Veracruz, Mexico, 90\nkm southeast of Veracruz city. This range of mountains lies in the northwestern portion of the Isthmus of Tehuantepec and is isolated from the Sierra Madre Oriental by extensive lowlands. Los Tuxtlas encompass approximately 4,200 km2, and the range is dominated by Volc\u00e1n San Mart\u00edn and Volc\u00e1n Santa Marta, each reaching more than 1,500 m elevation. The Gulf of Mexico lies a short distance from the mountains to the north and east. The northernmost Neotropical evergreen rainforest formerly dominated the habitat in the region (Andrle, 1966; Pennington & Sarukhan, 1968; Dirzo & Miranda, 1991), but due to deforestation it is now a mosaic with a high percentage of pasture, cropland, fencerows, and isolated trees (pers. obs.; Dirzo & Garcia 1992; Estrada, Coates-Estrada, & Merritt 1997). Andrle (1966) estimated that 50% of the region was forested in 1962. By 1975 Rappole & Warner (1980) estimated that a third of the forests still stood. Just 15% of forest remained in 1986 (Winker, Rappole, & Ramos, 1990; Dirzo & Garcia, 1992), and in 1994 only 7-10% of the region was forested (Winker, 1997). Remaining forest occurs primarily in the highlands, and below 500 m forest is scarce (Rappole, Powell, & Sader, 1994; Mendoza, Fay, & Dirzo, 2005; Figs. 1, 2).\nThe climate in Los Tuxtlas is warm and wet, with a mean annual\n102\n103\n104\n105\n106\n107\n108\n109\n110\n111\n112\n113\n114\n115\n116\n117\n118\n119\n120\n121\n122\n123\n124\n125\nPeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013)\nR ev ie w in g M an\nus cr ip t\ntemperature of 25 C, and annual precipitation is 4,500-4,900 mm, with a short dry season from March-May (Soto & Gama 1997). Canopy heights in primary forest range from 30-35 m (Ibarra-Manriquez et al., 1997). Second growth areas generally have variable canopy heights from 3-20 m (pers. obs.).\nIn 1967 the Universidad Nacional Aut\u00f3noma de M\u00e9xico established the\nEstaci\u00f3n de Biolog\u00eda Los Tuxtlas, protecting a 640-ha tract of lowland\nrainforest (18\u00b0 34\u201930\u201dN, 95\u00b0 04\u201920\u201dW; Gonz\u00e1lez-Soriano, Dirzo, & Vogt, 1997).\nOver the following decades this site became largely isolated from other tracts of forest, although a corridor of forest remains, connecting to the more extensive upland forests on Volc\u00e1n San Mart\u00edn (Dirzo & Garcia, 1992; Fig. 1). The first intensive sampling of birds in the region began in 1973, and data from that effort are included here (see Winker, 1997).\nDuring the non-breeding seasons of 1973-74 and 1974-75\nOehlenschlager, Ramos, Rappole, and Warner conducted the first intensive mist-netting efforts in the area. Sites extended through what was then contiguous rainforest from the biological station eastward to the coast (Fig. 3). In 1986, Rappole, Ramos, and Winker operated mist nets at the biological station, and Winker and Escalante continued work there from 1992 to 1994. In 2003-04 as part of a study of migrant birds, Shaw operated mist nets at the same location as Winker and Escalante\u2019s work in the 1990s. This study was approved by the University of Alaska Fairbanks IACUC (approval numbers: #00-33 & #04-03). Fieldwork occurred primarily during the non-breeding season. Effort was made to equally sample the available forest\n126\n127\n128\n129\n130\n131\n132\n133\n134\n135\n136\n137\n138\n139\n140\n141\n142\n143\n144\n145\n146\n147\n148\n149\nPeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013)\nR ev ie w in g M an\nus cr ip t\ntypes throughout the study period, although, in order to do this, habitat changes precluded using the same sites across all years (see Winker 1995; Fig. 3). Field effort as gauged by net hours also varied among years (Table 1).\nOur earliest sampling occurred over a wider area than later seasons\n(Fig. 3). During the earliest sampling, large tracts of contiguous forest consisting of various microhabitats dominated the region and were sampled accordingly (Fig. 3). This broader expanse of forest likely provided habitat to more species than the current distribution of forest. This increased detection probabilities for some species such as S. turdina, which was rare even during our earliest sampling. Two general types of forest were present after fragmentation: primary forest and acahual (second growth). Because our sampling was forest-oriented, our efforts tracked the distribution of these habitats. Primary rainforest and second growth habitats were sampled in all efforts. We were unable to separate capture data by site for the early sampling periods; our findings therefore include data from the somewhat larger area from the station east to the coast. This sampling heterogeneity leads us to be cautious and conservative in our analyses and interpretations. Importantly, however, the same site and net lanes were used in the 1992-2004 efforts (sample periods 4-8 in Table 1).\nOnly resident species were used in our analyses due to seasonal\nmigration and the high levels of variance in abundance this causes among obligate migrants. Changes in relative abundance were detected by comparing capture rates (birds per 1000 net hours) from each year of sampling. Species not captured in later sampling efforts and those with\n150\n151\n152\n153\n154\n155\n156\n157\n158\n159\n160\n161\n162\n163\n164\n165\n166\n167\n168\n169\n170\n171\n172\n173\nPeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013)\nR ev ie w in g M an\nus cr ip t\napparently declining or increasing rates of capture were selected for more detailed analyses (instead of applying statistical tests across all species). Presence/absence patterns and observational data (daily checklists in later years) were also considered to provide insight into changes in abundance in low-density species that did not have sufficient samples for statistical testing. Species were considered for examination for presence/absence if they had not been captured since at least 1986-87. Vagrants, defined as those rarely encountered species whose ranges do not normally include the Sierra de Los Tuxtlas, were excluded (Winker et al., 1992; Howell & Webb, 1995). Only first-time captures were used in statistical analyses. Simple linear regression was used to detect changes in abundance for selected species. We looked for newly appearing species using presence/absence netting, observational, and specimen data. Daily checklists were used to augment mist-net data as a check to determine whether absence from the mist-net data was indicative of reality.\nSpecies showing statistically significant declines and those not\ncaptured or observed in later sampling periods were categorized by preferred habitat (edge, forest, or semi-open), food preference (fruit/nectar or insects), elevational range, and whether Los Tuxtlas was at the periphery or core of its geographic range (Howell & Webb, 1995). These characteristics were used to assess whether certain traits of the species increased their vulnerability to local extirpation. RESULTS\nDuring this study we accumulated 165,083 net hours, equivalent to\n174\n175\n176\n177\n178\n179\n180\n181\n182\n183\n184\n185\n186\n187\n188\n189\n190\n191\n192\n193\n194\n195\n196\n197\nPeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013)\nR ev ie w in g M an\nus cr ip t\n37.7 net years if netting with a single net occurred twelve hours per day (Table 1). A species accumulation curve for a representative year (1992) with below-average net hours (12,605; mean = 20,220) showed that the avifauna was effectively fully sampled during most field seasons (Fig. S1, though in documenting a species\u2019 absence it is the among-season, aggregate sampling that is important). In total, 122 nonmigratory species were captured (Appendix).\nSeven species showed statistically significant declines during the\nsampling period: Phaethornis striilgularis, Xenops minutus, Glyphorynchus\nspirurus, Onychorhynchus coronatus, Myiobius sulphureipygius, Henicorhina leucosticta, and Eucometis penicillata (Table 2). Of these taxa, four were captured throughout the sampling period: P. striilgularis, X. minutus, E. penicillata, and H. leucosticta. G. spirurus was last captured in 1975, O. coronatus in 1986, and M. sulphureipygius in 1994, the last season of autumn netting. Four other species were captured in substantial numbers during early sampling periods but were not captured in later years: Lepidocolaptes souleyetii, Ornithion semiflavum, Leptopogon amaurocephalus, and Coereba flaveola (the latter may be an intratropical migrant in this region; Ramos, 1983); however, these species failed to show statistically significant declines in linear regression analyses, perhaps due to nonlinear declines. L. souleyetii was last captured in 1993-94, and the others were last captured in 1994-95. One species, Hylomanes momotula, was captured from 1986-1995 but not in the 1970s or in 2003-04. Though there were no captures in the 1970s, one individual was collected on 17 May 1974 a few km northeast of the station. A\n198\n199\n200\n201\n202\n203\n204\n205\n206\n207\n208\n209\n210\n211\n212\n213\n214\n215\n216\n217\n218\n219\n220\n221\nPeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013)\nR ev ie w in g M an\nus cr ip t\nsimilar pattern occurred in Anabacerthia variegaticeps, with captures occurring only in the 1990s. Only two species (Trogon collaris and Xiphorhynchus flavigaster) showed significant increases during the study period.\nPresence/absence mist-net capture data for low-density species not\ncaptured after 1986-87 could be interpreted as suggesting that an additional 23 taxa were extirpated during the study (Table 3). However, we know from observational data that not all of these species were absent. These taxa included rarely captured species that are too large for effective mist-net capture or that prefer the forest canopy (e.g., Micrastur ruficollis, Cotinga amabilis), mixed/open habitat specialists (e.g., Thraupis abbas and T. episcopus), a small-stream specialist (Chloroceryle aenea), and highland species (e.g., Myadestes unicolor) that are either not prone to capture in mist nets or at our site. Species such as Tityra inquisitor, both Thraupis tanagers, and others were known to be present on the site or nearby but were not captured in later sampling periods. Four species of hummingbirds are included in Table 3, but due to inconsistent capture probabilities of low-density hummingbird species and non-definitive observational data with respect to accurate identification, we provide no hypotheses regarding their possible extirpation or persistence at the site; further work focusing on these species is warranted. There were six other species not in Tables 2 or 3 in which mist net data alone might suggest declines or absences (Appendix) during the entire study but which were present throughout from observational data; netting is not an effective sampling tool for these taxa\n222\n223\n224\n225\n226\n227\n228\n229\n230\n231\n232\n233\n234\n235\n236\n237\n238\n239\n240\n241\n242\n243\n244\n245\nPeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013)\nR ev ie w in g M an\nus cr ip t\nbecause of body size or forest stratum occupied (e.g., Glaucidium brasilianum, Ciccaba virgata, and Celeus castaneus) or because forest understory is not preferred habitat (e.g., Pitangus sulphuratus, Myiozetetes similis, and Volatinia jacarina; Appendix). The first three of these species require more focused study to determine abundances and possible declines.\nFour lower-density species have likely been extirpated: Taraba major,\nFormicarius analis, Grallaria guatimalensis, and Schiffornis turdina (Table 3). One low-density species that might seem to have been extirpated from our data, Elaenia flavogaster, is likely an intratropical migrant here (pers. obs.; Howell & Webb, 1995; Table 3). Several species were captured only in later sampling periods (Appendix) but were observed or collected throughout, suggesting that there were no additions to the biological station\u2019s resident avifauna during the study.\nBased on all available data during the study (netting and observational\ndata), a minimum of 11 species of birds appear to have been extirpated from the biological station over the past three decades. This translates into an average loss of 3.7 species per decade or a local loss of 2.0% of the entire Los Tuxtlas avifauna (561 spp.; Schaldach & Escalante, 1997), 4.1% of the resident avifauna (269 spp.; Schaldach & Escalante, 1997), or 9.0% of the resident species captured in our study (122 spp.; Appendix). All 16 species showing significant declines or no longer present on the site prefer some degree of forest cover (Table 4). Three species are edge specialists: O. semiflavum, O. mexicanus, and C. flaveola. Twelve prefer closed canopy forest: P. striilgularis, H. momotula, X. minutus, G. spirurus, T. major, F. analis,\n246\n247\n248\n249\n250\n251\n252\n253\n254\n255\n256\n257\n258\n259\n260\n261\n262\n263\n264\n265\n266\n267\n268\n269\nPeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013)\nR ev ie w in g M an\nus cr ip t",
    "v1_text": "results : During this study we accumulated 165,083 net hours, equivalent to 37.7 net years if netting with a single net occurred twelve hours per day (Table 1). A species accumulation curve for a representative year (1992) with below-average net hours (12,605; mean = 20,220) showed that the avifauna was effectively fully sampled during most field seasons (Fig. S2, though in documenting a species\u2019 absence it is the amongseason, aggregate sampling that is important). In total, 122 nonmigratory species were captured (Appendix). Seven species showed statistically significant declines during the sampling period: Phaethornis striigularis, Xenops minutus, Glyphorynchus spirurus, Onychorhynchus coronatus, Myiobius sulphureipygius, Henicorhina leucosticta, and Eucometis penicillata (Table 2). Of these taxa, four were captured throughout the sampling period: P. striigularis, X. minutus, E. penicillata, and H. leucosticta. G. spirurus was last captured in 1975, O. coronatus in 1986, and M. sulphureipygius in 1994, the last season of autumn netting. Four other species were captured in substantial numbers during early sampling periods but were not captured in later years: Lepidocolaptes souleyetii, Ornithion semiflavum, Leptopogon amaurocephalus, and Coereba flaveola (the latter may be an intratropical migrant in this region; Ramos, 1983); however, these species failed to show statistically significant declines in linear regression analyses, perhaps due to nonlinear declines. L. souleyetii was last captured in 1993-94, and the others were last captured in 1994-95. One species, Hylomanes momotula, was captured from 1986-1995 but not in the 1970s or in 2003-04. Though there were no captures in the 1970s, one individual was collected on 17 May 1974 a few km northeast of the station. A similar pattern occurred in Anabacerthia variegaticeps, with captures occurring only in the 1990s. Only two species (Trogon collaris and Xiphorhynchus flavigaster) showed 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t significant increases during the study period. Presence/absence mist-net capture data for low-density species not captured after 1986-87 could be interpreted as suggesting that an additional 23 taxa were extirpated during the study (Table 3). However, we know from observational data that not all of these species were absent. These taxa included rarely captured species that are too large for effective mist-net capture or that prefer the forest canopy (e.g., Micrastur ruficollis, Cotinga amabilis), mixed/open habitat specialists (e.g., Thraupis abbas and T. episcopus), a small-stream specialist (Chloroceryle aenea), and highland species (e.g., Myadestes unicolor) that are either not prone to capture in mist nets or at our site. Species such as Tityra inquisitor, both Thraupis tanagers, and others were known to be present on the site or nearby but were not captured in later sampling periods. Four species of hummingbirds are included in Table 3, but due to inconsistent capture probabilities of low-density hummingbird species and non-definitive observational data with respect to accurate identification, we provide no hypotheses regarding their possible extirpation or persistence at the site; further work focusing on these species is warranted. There were six other species not in Tables 2 or 3 in which mist net data alone might suggest declines or absences (Appendix) during the entire study but which were present throughout from observational data; netting is not an effective sampling tool for these taxa because of body size or forest stratum occupied (e.g., Glaucidium brasilianum, Ciccaba virgata, and Celeus castaneus) or because forest understory is not preferred habitat (e.g., Pitangus sulphuratus, Myiozetetes similis, and Volatinia jacarina; Appendix). The first three of these species require more focused study to determine abundances and possible declines. Four lower-density species have likely been extirpated: Taraba major, Formicarius analis, Grallaria guatimalensis, and Schiffornis turdina (Table 3). One 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t low-density species that might seem to have been extirpated from our data, Elaenia flavogaster, is likely an intratropical migrant here (pers. obs.; Howell & Webb, 1995; Table 3). Several species were captured only in later sampling periods (Appendix) but were observed or collected throughout, suggesting that there were no additions to the biological station\u2019s resident avifauna during the study. Based on all available data during the study (netting and observational data), a minimum of 11 species of birds appear to have been extirpated from the biological station over the past three decades. This translates into an average loss of 3.7 species per decade or a local loss of 2.0% of the entire Los Tuxtlas avifauna (561 spp.; Schaldach & Escalante, 1997), 4.1% of the resident avifauna (269 spp.; Schaldach & Escalante, 1997), or 9.0% of the resident species captured in our study (122 spp.; Appendix). All 16 species showing significant declines or no longer present on the site prefer some degree of forest cover (Table 4). Three species are edge specialists: O. semiflavum, O. mexicanus, and C. flaveola. Eleven prefer closed canopy forest: P. striigularis, H. momotula, X. minutus, G. spirurus, F. analis, G. guatimalensis, L. amaurocephalus, M. sulphureipygius, S. turdina, H. leucosticta, and E. penicillata. T. major prefers primary forest edge, second growth, and riparian thickets, while L. souleyetii prefers semi-open or partly cleared forest. Eleven of 16, or 68.8%, of the species showing declines or extirpations in this study are insectivores, whereas among all species captured 41% are insectivores. This trend was not significant, however (G-test with Williams\u2019 correction, P > 0.1). The Sierra de Los Tuxtlas is the northernmost limit of the ranges of 13 of the 16 species showing declines. G. guatimalensis and H. leucosticta are the only species with a distribution extending substantially to the north and west of the study site. The field site is well within the elevational limits for all 16 species (Table 4). 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t The two species that significantly increased in abundance over the sample period (Table 4) both occur here at the core of their ranges, elevational distributions, and in their preferred forest habitat. T. collaris is a frugivore, and X. flavigaster is an insectivore. DISCUSSION Although the absence of a species is not a clear indication of extirpation, our sampling effort, despite its heterogeneity, does suggest that at minimum a species\u2019 absence indicates a decline. It is possible that some of the species now apparently gone from the station may persist in other, unsampled fragments. If the data presented here and our interpretations of them are accurate, the extirpation of species from the Estaci\u00f3n de Biolog\u00eda Los Tuxtlas has been ongoing since its isolation. Such an \u201cextinction debt\u201d is a recognized component of deforestation, and models of empirical data show that in birds this occurs across decades, but the species affected and the mechanisms of species loss remain poorly understood (Tilman et al., 1994; Ewers & Didham, 2003, Robinson & Sherry, 2012). Since 1973, 16 species susceptible to capture in mist nets have either become locally extirpated or are showing significant declines in abundance. The total number of losses and declines is undoubtedly higher than presented, because species not regularly captured in mist nets, such as large-bodied and canopy species, were not adequately surveyed. Species known to have been extirpated from Los Tuxtlas include Sarcoramphus papa, Harpia harpyja, and Ara macao. Patten, G\u00f3mez de Silva, & Smith-Patten (2010) also documented the extirpation of the latter two in Chiapas, Mexico. Many additional species have also been categorized as endangered or threatened in the Sierra de Los Tuxtlas (see Winker, 1997). Our estimate of the average rate of avian losses from the station of 3.7 species per decade may not be directly comparable to other studies due to differences in habitat and 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t sampling, but it is similar to the rate of loss observed at Barro Colorado Island by Robinson (1999) of 3.3 species per decade. Our estimate, however, includes only those taxa captured in mist nets, whereas Robinson\u2019s work included all species detected through observation. Of the eight species with data sufficient for statistical analysis that showed local extirpation, six were lost between 1992 and 2004 (on the same site), suggesting a continuing extirpation of species from the station. Bierregaard & Lovejoy (1988, 1989) found that as surrounding habitat was lost, species richness in remaining fragments increased as individuals displaced from surrounding areas found their way to remaining forest patches. This increased richness was limited by the lifespan of the individual birds (Bierregaard & Lovejoy 1988, 1989). Unlike these studies, in which forest patches were suddenly and completely isolated, the forest of the Estaci\u00f3n de Biolog\u00eda Los Tuxtlas was isolated gradually. Because extirpation seems to be continuing, we expect declines and extirpations to continue for some time at the station, even if no further deforestation occurs in the region (Willis, 1974; Brooks, Pimm, & Oyugi, 1999, Robinson, 1999; Ferraz et al., 2003). Mechanisms for tropical bird species losses due to deforestation and fragmentation probably include factors such as greater specialization as compared to temperate birds, reduced dispersal abilities, lower population densities, and patchy distributions (Robinson et al., 2004; Stratford & Robinson, 2005; Moore et al., 2008; Rompr\u00e9 et al., 2007). Our assessment of possible causes for the loss of these species reveals no definite patterns, however, other than the predominant requirement of forested habitat. On Barro Colorado Island in Lake Gat\u00fan, Panama, maturation of habitat and loss of open areas was responsible for the decline in the island\u2019s avifauna (Willis, 1974; Karr, 1982). This is unlikely to be the case in Los Tuxtlas. Despite major 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t degradation of surrounding forests, the station has remained primary forest with areas of second growth. A loss of sapling and seedling species has been described (Dirzo & Miranda, 1990), but the overall structure of the forest appears to have remained fairly stable. Vetter et al. (2011) found in a meta-analysis of 30 studies that the effects of fragmentation are not subject to simple generalities, and that they are highly site specific. Patten & Smith-Patten (2011) pointed to the need to understand extirpations at local scales because responses can differ from predictions made at larger scales. Los Tuxtlas is at the northernmost extent of the ranges of 13 of the 16 species we found to be declining or extirpated (Tables 3 and 4). Evidence is mixed as to whether populations at the periphery of a species\u2019 range are more vulnerable to extirpation (Terborgh & Winter, 1980; Kattan, Halvarez-Lopez, & Giraldo, 1994; Johnson, 1998). Los Tuxtlas is at the edge of all species\u2019 geographic ranges endemic to Neotropical rainforest, so it is not clear why this subset might be more subject to this phenomenon. The elevational distribution of each of these species encompasses sea level to 750 m or more (Howell & Webb, 1995), and we consider this factor unlikely to be responsible for the vulnerability of these particular taxa. Although insectivores showed a trend toward being disproportionately affected in our study, it was not significant. Elsewhere insectivores have been shown to be particularly vulnerable to severe habitat change (e.g., Kattan, Halvarez-Lopez, & Giraldo, 1994; Canaday, 1996; Johnson & Winker, 2010; Vetter et al., 2011). Additionally, deforestation can negatively impact species found in multi-species foraging flocks (Van Houtan et al., 2006), which are important to many birds of tropical rainforest communities (Willis, 1966; Morton, 1973; Buskirk, 1976; Rappole et al., 1983). Rappole & Morton (1985) noted that X. minutus, one of the species showing a significant decline in our study (Table 2) was a regular member of mixed flocks in the Sierra de Los Tuxtlas. 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t We considered large-scale range shifts, perhaps from climate change, as a possible cause for species loss, but this seems unlikely. At least some of the species lost in our study appear to have persisted in the southern portion of Los Tuxtlas near Volc\u00e1n Santa Marta at least into the mid-1990s (Winker, pers. obs.). If range shifts were the cause, species would likely have disappeared region-wide and we would not expect only forest-related species to be affected. Habitat loss and degradation seem to be the best explanations for the losses observed, but exactly how these changes affected each species remains unknown. Another possible influence on mist-net captures, particularly in the most recent, late winter/spring sampling periods, would be seasonal intra-tropical and elevational movements in some of the study species (Ramos, 1983, 1988). There is evidence that C. flaveola and E. flavogaster move seasonally within the tropics, seemingly to breed in Los Tuxtlas then departing (Ramos & Rappole, pers. obs.). Vega Rivera (1982) found probable elevational movements in M. sulphureipygius. The extirpations of seven of the 16 species are particularly notable. C. flaveola is a widely distributed species known to thrive in manipulated habitats such as gardens and forest edges and is a generalist frugivore and nectarivore (pers. obs.; Howell & Webb, 1995). This is not a species we would expect to decline due to forest fragmentation; both its habitat and food preferences are well suited to survival in a mosaic landscape, and it is known to persist in a fragmented landscape elsewhere in northern Middle America (Johnson & Winker, 2010). Intratropical migrations of C. flaveola may partially explain the changing capture rates in this species (Ramos & Rappole, pers. obs.). O. semiflavum and L. amaurocephalus are both edge specialists; thus, limited fragmentation, creating an increase in edges, might a priori seem to benefit these species. Though the habitat protected by the station has remained relatively static, the intensity of lowland 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t deforestation in Los Tuxtlas as a whole (Fig. 1) may be too extensive even for these edge specialists. L. souleyetii prefers open forest and partially cleared areas (Howell & Webb, 1995). The habitat surrounding the station during the 1980s and 1990s was dominated by pasture scattered with isolated trees. In our later field seasons there was a noticeable decline in the number of isolated trees and fences constructed of living trees (Winker, pers. obs.). This loss may account for the extirpation of L. souleyetii. G. spirurus apparently disappeared from the station between the 1970s and 1986, the first of the documented extirpations. The majority of deforestation across the region took place during this period. This previously abundant species disappeared from our data in just over a decade. Interestingly, on the slopes of neighboring Volc\u00e1n Santa Marta the species was present at least into the 1990s and probably still persists there (Winker, pers. obs.). Also, Estrada, Coates-Estrada, & Merritt (1997) had observational data of the species\u2019 presence in the station area in 1990-1992, indicating at least a decline if not extirpation (Table 2). In Brazil, G. spirurus persisted in experimentally isolated fragments well after isolation (Stouffer & Bierregaard 1995), and the species persists in highly fragmented forest in southern Belize (Johnson & Winker, 2010). H. momotula was collected but not netted in 1974, was captured in substantial numbers during 1986 and 1992-94, but was absent in the last two seasons of sampling. This pattern is mysterious. This species has an elevational range extending to 1500 m and may persist in the forests of the upper slopes of Volc\u00e1n San Mart\u00edn. If so, we speculate that the station may serve as a sink for this species, where habitat is insufficient for a self-sustaining population but may occasionally be colonized by dispersing individuals (see also Winker et al., 1997). Continued sampling may provide more insight into its abundance patterns. It illustrates the need for improved understanding of species-specific dispersal behavior within and among forest fragments (e.g., Van Houtan et al., 2007; Moore et al., 2008; Ibarra353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t Macias, Robinson, & Gaines 2011), which may be an important driver for patterns such as those we observed. Two other studies provide comparative value to our results. The four species we consider likely extirpated (Taraba major, Formicarius analis, Grallaria guatimalensis, and Schiffornis turdina) were not detected in the much broader census surveys of Estrada, Coates-Estrada, & Merritt (1997) in 1990-1992. Patten, G\u00f3mez de Silva, & Smith-Patten (2010) conducted the geographically closest long-term study to ours in their analysis of avian declines at Palenque, Chiapas, Mexico. Their results showed only three species that overlapped our results. They found Eucometis penicillata extirpated (to our decline) and two others that declined as our populations did (Xenops minutus and Leptopogon amaurocephalus). Indeed, the species-level heterogeneity between our studies is noteworthy. A key similarity between our studies, however, is the importance of forest in explaining declines and extripations (Patten & Smith-Patten, 2011). Our analyses suggest that the Estaci\u00f3n de Biolog\u00eda Tropical Los Tuxtlas is too small to maintain its full, historic complement of bird species. If deforestation accelerated region-wide, eliminating other forest refugia, the station alone (640 ha) would be unable to maintain the historical avian diversity of the region or to provide source populations for restored forest habitats for many of its present bird species. Given the scale of deforestation in the region, it is surprising that there are not more species showing declines. Indeed, we may consider it good news that important forest seed dispersers such as Habia tanagers (Puebla & Winker, 2004) did not show significant declines. The overall size of the remaining forests, particularly in the highlands, may be ameliorating the effects of lowland deforestation. However, increasing or continued isolation of the station will probably limit recolonization from elsewhere, and species losses will likely continue. 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t In our study, although several species seemed to quickly succumb to local and regional deforestation, others showed delayed declines and extirpations, a phenomenon also known to happen at larger scales (Tilman et al., 1994, Pimm et al., 2006). Moreover, the effects of deforestation were remarkably heterogeneous among forest-related species, with no single clear pattern of why some species experienced declines or extirpation. Our long-term data suggest that predicting which species will be most affected by deforestation in the northern Neotropics, and thus effectively working to ameliorate the effects of forest loss, will be particularly challenging. Nevertheless, as similar long-term datasets accrue, subtle patterns may reveal how species-specific responses reflect underlying commonalities that can be exploited for effective management and conservation. ACKNOWLEDGMENTS We thank the many field assistants who have helped us over the years and those who have provided and helped obtain permits to conduct this research. R. Barry, A. Powell, S. Pimm, M. Patten, and three anonymous reviewers provided excellent advice and comments. LITERATURE CITED Andr\u00e9n H. 1994. Effects of habitat fragmentation on birds and mammals in landscapes with different proportions of suitable habitat: a review. Oikos 71:355-366. Andrle RF. 1966. North American migrants in the Sierra de Tuxtla of southern Veracruz, Mexico. Condor 68:177-184. Barlow J, Peres CA, Henriques LMP, Stouffer PC, Wunderle JM. 2006. The responses of understorey birds to forest fragmentation, logging and wildfires: An Amazonian synthesis. Biological Conservation 128:182-192. Becker P, Moure JS, Peralta FJA. 1991. More about euglossine bees in 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t Amazonian forest fragments. Biotropica 23:586-591. Bierregaard RO, Lovejoy TE. 1988. Birds in Amazonian forest fragments: Effects of insularization. Pages 1564-1579 in: Acta XIX Congressus Internationalis Ornithologici (H. Oellet, ed.). University of Ottawa Press, Ottawa, Canada. Bierregaard RO, Lovejoy TE. 1989. Effects of forest fragmentation on Amazonian understory bird communities. Acta Amazonica 19:215-241. Blake JG. 1991. Nested subsets and the distribution of birds in isolated woodlots. Conservation Biology 5:58-86. Brook BW, Sodhi NS, Ng PKL. 2003. Catastrophic extinctions follow deforestation in Singapore. Nature 424:420-423. Brooks TM, Pimm SL, Oyugi JO. 1999. Time lag between deforestation and bird extinction in tropical forest fragments. Conservation Biology 13:1140-1150. Buskirk WH. 1976. Social systems in a tropical forest avifauna. American Naturalist 110:293-310. Canaday C. 1996. Loss of insectivorous birds among a gradient of human impact in Amazonia. Biological Conservation 77:63-77. Ceballos G, Ehrlich PR. 2002. Mammal population losses and the extinction crisis. Science 296:904-907. Daily GC, Ehrlich PR. 1995. Preservation of biodiversity in small rainforest patches: rapid evaluations using butterfly trapping. Biodiversity and Conservation 4: 35-55. Dirzo R, Garcia MC. 1992. Rates of deforestation in Los Tuxtlas, a Neotropical area in southeast Mexico. Conservation Biology 6:84-90. Dirzo R, Miranda A. 1990. Contemporary Neotropical defaunation and forest structure, function, and diversity, a sequel to John Terborgh. Conservation Biology 4:444- 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t 447. Dirzo R, Miranda A. 1991. Altered patterns of herbivory and diversity in the forest understory: a case study of the possible consequences of contemporary defaunation. Pages 273-287 in: Plant-Animal Interactions: Evolutionary ecology in tropical and temperate regions (Price PW, Lewinsohn TM, Fernandes GW, Benson WW, eds.). Wiley and Sons, Inc., New York. Dirzo R, Raven PH. 2003. Global state of biodiversity and loss. Annual Review of Environment and Resources 28:137-167. Estrada A, Coates-Estrada R, Merritt Jr DA. 1997. Anthropogenic landscape changes and avian diversity at Los Tuxtlas, Mexico. Biodiversity and Conservation 6:19-43. Ewers RM, Didham RK. 2006. Confounding factors in the detection of species responses to fragmentation. Biological Reviews 81:117-142. Faaborg J, Brittingham M, Donovan T, Blake J. 1995. Habitat fragmentation in the temperate zone. Pages 357-380 in: Ecology and management of Neotropical migratory birds: a synthesis and review of the critical issues (Finch DM, Martin TE, eds.). Oxford University Press, Cambridge, United Kingdom. Fahrig L. 2003. Effects of habitat fragmentation on biodiversity. Annual Review of Ecology Evolution and Systematics 34:487-515. Ferraz G, Russell GJ, Stouffer PC, Bierregaard RO, Pimm SL, Lovejoy TE. 2003. Rates of species loss from Amazonian forest fragments. Proceedings of the National Academy of Sciences USA 100:14069-14073. Ferraz G, Nichols JD, Hines JE, Stouffer PC, Bierregaard RO, Lovejoy TE. 2007. A large- scale deforestation experiment: Effects of patch area and isolation on Amazon birds. Science 315:238-241. Gonz\u00e1lez-Soriano E, Dirzo R, Vogt RC, eds. 1997. Historia Natural de Los Tuxtlas. 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t Universidad Nacional Aut\u00f3noma de M\u00e9xico, M\u00e9xico, D.F. Howell S, Webb S. 1995. A guide to the birds of Mexico and northern Central America. Oxford University Press, New York. Ibarra-Macias A, Robinson WD, Gaines MS. 2011. Experimental evaluation of bird movements in a fragmented Neotropical landscape. Biological Conservation 144:703-712. Ibarra-Manr\u00edquez G, Martinez-Ramos M, Dirzon R, N\u00fa\u00f1ez-Farf\u00e1n J. 1997 La vegetaci\u00f3n. Pages 61\u201374 in Historia Natural de Los Tuxtlas (Gonz\u00e1lez-Soriano E, Dirzo R, Vogt RC, Eds). Universidad Nacional Aut\u00f3noma de M\u00e9xico, M\u00e9xico, D.F. Johnson AB, Winker K. 2010. Short-term hurricane impacts on a Neotropical community of marked birds. PLoS ONE 5:e15109. Johnson CN. 1998. Species extinction and the relationship between distribution and abundance. Nature 394:272-274. Kattan GH, Halvarez-Lopez H, Giraldo M. 1994. Forest fragmentation and bird extinctions: San Antonio eighty years later. Conservation Biology 8:138-146. Karr JR. 1982. Population variability and extinction in a tropical land-bridge island. Ecology 63:1975-1978. Laurance WF, et al. 2011. The fate of Amazonian forest fragments: A 32-year investigation. Biological Conservation 144:56-67. Leck CF. 1979. Avian extinctions in an isolated tropical wet-forest preserve, Ecuador. Auk 96:343-352. Lees AC, Peres CA. 2006. Rapid avifaunal collapse along the Amazonian deforestation frontier. Biological Conservation 133:198-211. Leigh EG, Jr. 1975. Population fluctuations, community stability, and environmental variability. Pages 52-73 in: Ecology and evolution of communities (Cody ML, 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t Diamond JM, eds.). Belknap Press, Cambridge, Massachusetts. Leigh EG, Jr. 1981. The average lifetime of a population in a varying environment. Journal of Theoretical Biology 90:213-239. Lovejoy TE, Rankin JM, Bierregaard RO, Brown KS, Jr., Emmons LH, Van Der Voort ME. 1984. Ecosystem decay of Amazon forest remnants. Pages 295-325 in: Extinctions (Nitecki MH, ed.). University of Chicago Press, Chicago, Illinois. Lovejoy TE, Bierregaard RO, Rylands AB, Malcolm JR, Quintela CE, Harper LH, Brown KS, Powell AH, Powell GVN, Schubart HOR, Hays MB. 1986. Edge and other effects of isolation on Amazon forest fragments. Pages 257-285 in: Conservation biology: The science of scarcity of diversity (Soule ME, ed.). Sinauer Associates, Sunderland, Massachusetts. Malcolm JR. 1988. Small mammal abundances in isolated and non-isolated primary forest reserves near Manaus, Brazil. Acta Amazonica 18: 67-83. Mendoza E, Fay J, Dirzo R. 2005. A quantitative analysis of forest fragmentation in Los Tuxtlas, southeast Mexico: patterns and implications for conservation. Revista Chilena de Historia Natural 78:451-467. Moore RP, Robinson WD, Lovette IJ, Robinson TR. 2008. Experimental evidence for extreme dispersal limitation in tropical forest birds. Ecology Letters 11: 960-968. Morton ES. 1973. On the evolutionary advantages and disadvantages of fruit eating in tropical birds. American Naturalist 107:8-22. Nemark WD. 1991. Tropical forest fragmentation and the local extinction of understory birds in the eastern Usambara Mountains, Tanzania. Conservation Biology 5:67- 78. O\u2019Grady JJ, Reed DH, Brook BW, Frankham R. 2004. What are the best correlates of predicted extinction risk? Biological Conservation 118:513-520. 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t Pahl LI, Winter JW, Heinsohn G. 1988. Variation in responses of arboreal marsupials to fragmentation of tropical rainforest in north eastern Australia. Biological Conservation 46:71-82 Patten MA, Gomez de Silva H, Smith-Patten BD. 2010. Long-term changes in the bird community of Palenque, Chiapas, in response to rainforest loss. Biodiversity and Conservation 19:21-36. Patten MA, Smith-Patten BD. 2011. Predictors of occupancy trend across spatial scale. Conservation Biology 6:1203-1211. Pennington TD, Sarukhan J. 1968. Arboles Tropicales de Mexico. Instituto Nacional de Investigacciones Forestales, D.F., Mexico. Pimm SL, Jones HH, Diamond J. 1988. On the risk of extinction. American Naturalist 132:757-785. Pimm SL, Raven P, Peterson A, \u015eekercio\u011flu \u00c7, Ehrlich PR. 2006. Human impacts on the rates of recent, present, and future bird extinctions. Proceedings of the National Academy of Sciences USA 103:10941-10946. Powell AH, Powell GVN. 1987. Population dynamics of male euglossine bees in Amazonian forest fragments. Biotropica 19: 176-179. Puebla F, Winker K. 2004. Dieta y dispersi\u00f3n de semillas de dos especies de tangara (Habia) en dos tipos de vegetaci\u00f3n en Los Tuxtlas, Veracruz, M\u00e9xico. Ornitolog\u00eda Neotropical 15:53-64. Ramos MA. 1983. Seasonal movements of bird populations at a Neotropical study site in southern Veracruz, Mexico. Ph.D. dissertation. University of Minnesota, Minneapolis. Ramos MA. 1988. Eco-evolutionary aspects of bird movements in the northern Neotropical region. Pages 251-293 in: Acta XIX Congressus Internationalis 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t Ornithologici, Vol. I, (Ouellet H, ed.). University of Ottawa Press, Ottawa, Canada. Rappole JH, Warner DW. 1980. Ecological aspects of migrant bird behavior in Veracruz, Mexcio. Pages 353-393 in: Migrant Birds in the Neotropics: Ecology, Behavior, Distribution, and Conservation, (Keast A, Morton ES, eds.). Smithsonian Institution Press, Washington, D. C. Rappole JH, Morton ES, Lovejoy TE, III, Ruos JL. 1983. Nearctic avian migrants in the Neotropics. United States Fish and Wildlife Service, Washington D.C. Rappole JH, Powell GVN, Sader SA. 1994. Remote-sensing assessment of tropical habitat availability for a Nearctic migrant: the Wood Thrush. Pages 91-103 in: Mapping the diversity of nature (Miller RI, ed.). Chapman and Hall, London. Rappole JH, Winker K, Powell GVN. 1998. Migratory bird habitat use in southern Mexico: mist nets versus point counts. Journal of Field Ornithology 69:635-643. Remsen JV Jr., Good DA. 1996. Misuse of data from mist-net captures to assess relative abundance in bird populations. Auk 113: 381-398. Robbins CS. 1980. Effect of forest fragmentation on breeding bird populations in the piedmont of the mid-Atlantic region. Atlantic Naturalist 33:31-36. Robinson WD. 1999. Long-term changes in the avifauna of Barro Colorado Island, Panama, a tropical forest isolate. Conservation Biology 13:85-97. Robinson WD, Sherry TW. 2012. Mechanisms of avian population decline and species loss in tropical forest fragments. Journal of Ornithology 153:S141-S152. Robinson WD, Angehr GR, Robinson TR, Petit LJ, Petit DR, Brawn JD. 2004. Distribution of bird diversity in a vulnerable Neotropical landscape. Conservation Biology 18:510-518. Rolstad J. 1991. Consequences of forest fragmentation for the dynamics of bird 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t populations: conceptual issues and the evidence. Biological Journal of the Linnean Society 42:149-163. Rompr\u00e9 G, Robinson WD, Desrochers A, Angehr G. 2007. Environmental correlates of avian diversity in lowland Panama rainforests. Journal of Biogeography 34:802- 815. Schaldach W, Escalante P. 1997. Lista de Aves. Pages 571-573 in: Historia Natural de Los Tuxtlas (Gonz\u00e1lez-Soriano E, Dirzo R, Vogt RC, eds.). Universidad Nacional Autonoma de Mexico. D.F., Mexico. Sodhi NS, Liow LH, Bazzaz FA. 2004. Avian extinctions from tropical and subtropical forests. Annual Review of Ecology Evolution and Systematics 35:323-345. Soto M, Gama L. 1997. Climas. Pages 7-23 In: Historia Natural de Los Tuxtlas (Gonz\u00e1lez-Soriano E, Dirzo R, Vogt RC, eds.). Universidad Nacional Autonoma de Mexico. D.F., Mexico. Stotz DF, Fitzpatrick JW, Parker TA, Moskovits DK. 1996. Neotropical birds: ecology and conservation. University of Chicago Press, Chicago. Stouffer PC, Bierregaard RO. 1995. Effects of forest fragmentation on understory insectivorous birds. Ecology 76:2429-45. Stratford JA, Robinson WD. 2005. Gulliver travels to the fragmented tropics: geographic variation in mechanisms of avian extinction. Frontiers in Ecology and the Environment 3:85-92. Stuart SN, Chanson JS, Cox NA, Young BE, Rodrigues ASL, Fischman DL, Waller RW. 2004. Status and trends of amphibian declines and extinctions worldwide. Science 306:1783-1786. Terborgh J, Winter B. 1980. Some causes of extinction. Pages 119-133 In: Conservation biology: an evolutionary-ecological perspective (Soule ME, Wilcox BA, eds.). 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t Sinauer Associates, Sunderland, Massachusetts. Tilman D, May RM, Lehman CL, Nowak MA. 1994. Habitat destruction and the extinction debt. Nature 371:65-66. Turner IM. 1996. Species loss in fragments of tropical rain forest: a review of the evidence. Journal of Applied Ecology 33:200-209. Van Houtan KS, Pimm SL, Bierregaard RO, Jr., Lovejoy TE, Stouffer PC. 2006. Local extinctions in flocking birds in Amazonian forest fragments. Evolutionary Ecology Research 8:129-148. Van Houtan KS, Pimm SL, Halley JM, Bierregaard RO Jr., Lovejoy TE. 2007. Dispersal of Amazonian birds in continuous and fragmented forest. Ecology Letters 10:219- 229. Vega Rivera JH. 1982. Aspectos biologicos de Myiobius sulphureipygius (Aves: Tyrannidae) en el area de Santa Marta region de \u201cLos Tuxtlas,\u201d Veracruz, Mexico. Professional thesis. Universidad Nacional Autonoma de Mexico. Vetter D, Hansbauer MM, V\u00e9gv\u00e1ri Z, Storch I. 2011. Predictors of forest fragmentation sensitivity in Neotropical vertebrates: a quantitative review. Ecography 34:1-8. Whitman AA, Hagan JM III, Brokaw NVL. 1997. A comparison of two survey techniques used in a subtropical forest. Condor 99:955-965. Willis EO. 1966. The role of migrant birds at swarms of army ants. Living Bird 5:187- 231. Willis EO. 1974. Populations and local extinctions of birds on Barro Colorado Island, Panama. Ecological Monographs 44:153-169. Willis EO. 1979. The composition of avian communities in remanescent woodlots in southern Brazil. Papeis Avulsos de Zoolgia 33:1-25. Winker K. 1995. Habitat selection in woodland Nearctic-Neotropic migrants on the 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t Isthmus of Tehuantepec. I. Autumn migration. Wilson Bulletin 107:26-39. Winker K. 1997. Introduction to the birds of Los Tuxtlas. Pages 535-541 in: Historia Natural de Los Tuxtlas (Gonz\u00e1lez-Soriano E, Dirzo R, Vogt RC, Eds.). Universidad Nacional Autonoma de Mexico. D.F., Mexico. Winker K, Rappole JH, Ramos MA. 1990. Population dynamics of the Wood Thrush (Hylocichla mustelina) on its wintering grounds in southern Veracruz, Mexico. Condor 92:444-460. Winker K, Oehlenschlager RJ, Ramos MA, Zink RM, Rappole JH, Warner DW. 1992. Avian distribution and abundance records for the Sierra de Los Tuxtlas, Veracruz, Mexico. Wilson Bulletin 104:699-718. Winker K, Escalante P, Rappole JH, Ramos MA, Oehlenschlager RJ, Warner DW. 1997. Periodic migration and lowland forest refugia in a sedentary Neotropical bird, Wetmore\u2019s bush-tanager. Conservation Biology 11:692-697. Zimmerman BL, Bierregaard RO. 1986. Relevance of the equilibrium theory of island biogeography and species-area relations to conservation with a case from Amazonia. Journal of Biogeography 13:133-143. 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t figure captions : Figure 1. Comparative views of the Sierra de Los Tuxtlas from an artificially colorized 1979 Landsat image (top) and a 2010/11 Google Earth image (bottom) showing the extent of deforestation in the region. Remaining forest has become concentrated at higher elevations on the slopes of the region\u2019s three volcanoes, San Mart\u00edn, Santa Marta, and San Mart\u00edn Paj\u00e1pan (the forested areas remaining, from left to right). Figure 2. Satellite view of Volc\u00e1n San Mart\u00edn, the northernmost volcano in the Sierra de Los Tuxtlas, showing the distribution of forests (dark areas). The study area is indicated by the white box, which corresponds to the area in Fig. 3 (image from Google Earth, 2010). Figure 3. Maps of the study area in the northern lowlands of the Sierra de Los Tuxtlas (this is the area in the white box in Fig. 2) showing a rough outline of all forests types (dark gray areas) in 1979 (top, from Landsat image), in 2005 (bottom, from GoogleEarth), and netting sites (black polygons). Numbers indicate field season(s) site was used and correspond to rows in Table 1. Figure S1. A series of satellite images depicting deforestation in Los Tuxtlas, focusing in on the volcanoes Santa Marta (left) and San Mart\u00edn Paj\u00e1pan (right), starting with a 1973/4 Skylab image (upper left) and progressing through a series of Landsat images, from 1999 (upper right), 2003 (lower left), and 2011 (lower right). Figure S2. Species accumulation curve for a representative year with below average net 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t hours (1992, 12,605 net hours).667 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t Table 1(on next page) Sample effort and periods during eight nonbreeding seasons across three decades in the Sierra de Los Tuxtlas, Veracruz, Mexico. Sample effort and periods during eight nonbreeding seasons across three decades in the species f p r2 last captured : Phaethornis striigularisc 6.337 0.045 0.514 2002-03 Hylomanes momotulaa 0.210 0.890 0.003 1994-95 Trogon collarisb 7.041 0.038 0.540 n/a Xiphorhynchus flavigasterb 6.941 0.039 0.536 n/a Xenops minutusc 7.578 0.033 0.558 2003-04 Glyphorynchus spirurusc,d 7.529 0.034 0.557 1974-75 Lepidocolaptes souleyetiid 3.265 0.121 0.352 1992-93 Ornithion semiflavumd 0.327 0.588 0.052 1994-95 Leptopogon amaurocephalusd 2.814 0.144 0.319 1994-95 Onychorhynchus coronatusc,d 6.861 0.040 0.533 1986-87 Myiobius sulphureipygiusc,d 10.555 0.019 0.629 1994-95 Henicorhina leucostictac,d 6.740 0.041 0.529 2003-04 Coereba flaveolad 2.164 0.192 0.265 1994-95 Eucometis penicillatac 18.725 0.005 0.757 2002-03 a Species captured 1986-1995. See text. 1 2 3 4 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t b Species showing an increase in abundance. c Species showing a significant decline. d Species not captured in later sampling periods. 5 6 7 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t Table 3(on next page) Species not captured or observed from 1992-2004, seasons captured (from Appendix), presence on the field site in later sampling periods, and comments. Species not captured or observed from 1992-2004, seasons captured (from Appendix), presence on the field site in later sampling periods, and comments. PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t Table 3. Species not captured or observed from 1992-2004, seasons captured (from Appendix), presence on the field site in later sampling periods, and comments. Micrastur ruficollis 1 Y observed Crypturellus boucardi 3 Y observed Heliomaster longirostris 1 ? hummingbird Florisuga mellivora 1 ? hummingbird Chlorostilibon canivetii 2 ? hummingbird Hylocharis eliciae 1, 2 ? hummingbird Chloroceryle aenea 1, 2 Y small streams Dryocopus lineatus 2 Y observed Synallaxis erythrothorax 2 Y observed Taraba major 2 N forest understory Formicarius analis 1 N forest understory Grallaria guatimalensis 1, 3 N forest understory Tityra inquisitor 1 Y observed, canopy Cotinga amabilis 1 ? canopy Schiffornis turdina 1 N forest understory Polioptila plumbea 1 Y observed Myadestes unicolor 1 Y highlands Euphonia affinis 2 ? none 1 2 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t Thraupis abbas 1 Y observed Thraupis episcopus 2 Y observed Saltator atriceps 1, 2 Y observed Molothrus aeneus 1 Y observed PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t Table 4(on next page) Habitat, foraging preference, elevational range, and position within geographical distribution for 18 species of birds at the Estaci\u00f3n de Biolog\u00eda Los Tuxtlas (from Howell and Webb, 1995). Habitat, foraging preference, elevational range, and position within geographical distribution for 18 species of birds at the Estaci\u00f3n de Biolog\u00eda Los Tuxtlas (from Howell and Webb, 1995). PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t Table 4. Habitat, foraging preference, elevational range, and position within geographical distribution for 18 species of birds at the Estaci\u00f3n de Biolog\u00eda Los Tuxtlas (from Howell and Webb, 1995). Habitat Foraging Elevational Geographic Species preference guild distribution (m) distribution Phaethornis striigularis forest nectarivore 0-1500 periphery Hylomanes momotula forest frugivore 0-1500 periphery Trogon collaris forest frugivore 0-2400 core Xenops minutus forest insectivore 0-1000 periphery Xiphorhynchus flavigaster forest insectivore 0-1500 core Glyphorynchus spirurus* forest insectivore 0-1200 periphery Lepidocolaptes souleyetii semi-open insectivore 0-1500 periphery Taraba major* forest insectivore 0-1600 periphery Formicarius analis* forest insectivore 0-750 periphery Grallaria guatimalensis* forest insectivore 50-3500 core Ornithion semiflavum edge insectivore 0-1500 periphery Leptopogon amaurocephalus edge insectivore 0-1300 periphery Onychorhynchus coronatus forest insectivore 0-1200 periphery Myiobius sulphureipygius forest insectivore 0-1000 periphery Schiffornis turdina* forest frugivore 0-750 periphery Henicorhina leucosticta forest insectivore 0-1300 core Coereba flaveola edge frugivore 0-1000 periphery Eucometis penicillata forest frugivore 0-750 periphery 1 2 3 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t * Presence/Absence data suggest species is extirpated.4 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t Figure 1 Comparative views of the Sierra de Los Tuxtlas from an artificially colorized 1979 Landsat image (top) and a 2010/11 Google Earth image (bottom) showing the extent of deforestation in the region. Remaining forest has become concentrated at higher elevatio Comparative views of the Sierra de Los Tuxtlas from an artificially colorized 1979 Landsat image (top) and a 2010/11 Google Earth image (bottom) showing the extent of deforestation in the region. Remaining forest has become concentrated at higher elevations on the slopes of the region\u2019s three volcanoes, San Mart\u00edn, Santa Marta, and San Mart\u00edn Paj\u00e1pan (the forested areas remaining, from left to right). PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t Figure 2 Satellite view of Volc\u00e1n San Mart\u00edn, the northernmost volcano in the Sierra de Los Tuxtlas, showing the distribution of forests (dark areas). The study area is indicated by the white box, which corresponds to the area in Fig. 3 (image from Google Earth, 2 Satellite view of Volc\u00e1n San Mart\u00edn, the northernmost volcano in the Sierra de Los Tuxtlas, showing the distribution of forests (dark areas). The study area is indicated by the white box, which corresponds to the area in Fig. 3 (image from Google Earth, 2010). PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t Figure 3 Maps of the study area in the northern lowlands of the Sierra de Los Tuxtlas (this is the area in the white box in Fig. 2) showing a rough outline of all forests types (dark gray areas) in 1979 (top, from Landsat image), in 2005 (bottom, from GoogleEarth) Maps of the study area in the northern lowlands of the Sierra de Los Tuxtlas (this is the area in the white box in Fig. 2) showing a rough outline of all forests types (dark gray areas) in 1979 (top, from Landsat image), in 2005 (bottom, from GoogleEarth), and netting sites (black polygons). Numbers indicate field season(s) site was used and correspond to rows in Table 1. PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t sierra de los tuxtlas, veracruz, mexico. : PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t Table 1. Sample effort and periods during eight nonbreeding seasons across three decades in the Sierra de Los Tuxtlas, Veracruz, Mexico. Nonbreeding Net Sampling season hours period 1) 1973-74 33,976 15 Aug-26 May 2) 1974-75 36,512 7 Aug-29 May 3) 1986-87 4,310 17 Nov-16 Jan 4) 1992-93 12,605 5 Sep-15 Nov 5) 1993-94 41,142 25 Aug-20 May 6) 1994-95 22,509 15 Aug-15 Nov 7) 2002-03 8,395 21 Feb-27 Apr 8) 2003-04 2,312 5 Apr-29 Apr 2 3 PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t Table 2(on next page) Outcomes of regression analyses for 14 species showing changes in abundance (capture rates; captures and rates are given in the Appendix) and those not detected in the later sampling periods. Those P-values presented in bold are significant at alpha = 0.0 Outcomes of regression analyses for 14 species showing changes in abundance (capture rates; captures and rates are given in the Appendix) and those not detected in the later sampling periods. Those P-values presented in bold are significant at alpha = 0.05. PeerJ reviewing PDF | (v2013:07:699:1:0:NEW 12 Sep 2013) R ev ie w in g M an us cr ip t Table 2. Outcomes of regression analyses for 14 species showing changes in abundance (capture rates; captures and rates are given in the Appendix) and those not detected in the later sampling periods. Those P-values presented in bold are significant at \u03b1 = 0.05.",
    "v2_text": "figure captions : Figure 1. Satellite view of Volc\u00e1n San Mart\u00edn, the northernmost volcano in the Sierra de Los Tuxtlas, showing the distribution of forests (dark areas). The study area is indicated by the white arrow (image from GoogleEarth, 2005). Figure 2. Comparative views of Volc\u00e1n Santa Marta and San Martin Pajapan in the southern Sierra de Los Tuxtlas from 1973 (top; NASA/Skylab) and 2005 (bottom; GoogleEarth) showing extent of deforestation, particularly severe in the lowlands. Figure 3. Maps of the study area in the northern lowlands of the Sierra de Los Tuxtlas showing a rough outline of all forests types (dark gray areas) in 1979 (top, from Landsat image), in 2005 (bottom, from GoogleEarth), and netting sites (black polygons). Numbers indicate field season(s) site was used and correspond to rows in Table 1. Figure S1. Species accumulation curve for a representative year with below average net hours (1992, 12,605 net hours). 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t Fig. 1 734 735 736 737 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t 738 739 740 741 742 R ev ie w in g M an us cr ip t 744 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t 747 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t Table 1(on next page) Tables 1 through 4 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t Table 1. Sample effort and periods during eight nonbreeding seasons across three decades in the Sierra de Los Tuxtlas, Veracruz, Mexico. Nonbreeding Net Sampling season hours period 1) 1973-74 33,976 15 Aug-26 May 2) 1974-75 36,512 7 Aug-29 May 3) 1986-87 4,310 17 Nov-16 Jan 4) 1992-93 12,605 5 Sep-15 Nov 5) 1993-94 41,142 25 Aug-20 May 6) 1994-95 22,509 15 Aug-15 Nov 7) 2002-03 8,395 21 Feb-27 Apr 8) 2003-04 2,312 5 Apr-29 Apr PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t Table 2. Outcomes of regression analyses for 14 species showing changes in abundance (capture rates; captures and rates are given in the Appendix) and those not detected in the later sampling periods. Those P-values presented in bold are significant at \u03b1 = 0.05. Species F P R 2 Last captured Phaethornis striilgularis c 6.337 0.045 0.514 2002-03 Hylomanes momotula a 0.210 0.890 0.003 1994-95 Trogon collaris b 7.041 0.038 0.540 n/a Xiphorhynchus flavigaster b 6.941 0.039 0.536 n/a Xenops minutus c 7.578 0.033 0.558 2003-04 Glyphorynchus spirurus c,d 7.529 0.034 0.557 1974-75 Lepidocolaptes souleyetii d 3.265 0.121 0.352 1992-93 Ornithion semiflavum d 0.327 0.588 0.052 1994-95 Leptopogon amaurocephalus d 2.814 0.144 0.319 1994-95 Onychorhynchus coronatus c,d 6.861 0.040 0.533 1986-87 Myiobius sulphureipygius c,d 10.555 0.019 0.629 1994-95 Henicorhina leucosticta c,d 6.740 0.041 0.529 2003-04 Coereba flaveola d 2.164 0.192 0.265 1994-95 Eucometis penicillata c 18.725 0.005 0.757 2002-03 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t a Species captured 1986-1995. See text. b Species showing an increase in abundance. c Species showing a significant decline. d Species not captured in later sampling periods. PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t Table 3. Species not captured or observed from 1992-2004, seasons captured (from Appendix), presence on the field site in later sampling periods, and comments. Species Seasons Captured Presence Comments Micrastur ruficollis 1 Y observed Crypturellus boucardi 3 Y observed Heliomaster longirostris 1 ? hummingbird Florisuga mellivora 1 ? hummingbird Chlorostilibon canivetii 2 ? hummingbird Hylocharis eliciae 1, 2 ? hummingbird Chloroceryle aenea 1, 2 Y small streams Dryocopus lineatus 2 Y observed Synallaxis erythrothorax 2 Y observed Taraba major 2 N forest understory Formicarius analis 1 N forest understory Grallaria guatimalensis 1, 3 N forest understory Tityra inquisitor 1 Y observed, canopy Cotinga amabilis 1 ? canopy Schiffornis turdina 1 N forest understory Polioptila plumbea 1 Y observed Myadestes unicolor 1 Y highlands Euphonia affinis 2 ? none Thraupis abbas 1 Y observed Thraupis episcopus 2 Y observed Saltator atriceps 1, 2 Y observed Molothrus aeneus 1 Y observed PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t Table 4. Habitat, foraging preference, elevational range, and position within geographical distribution for 18 species of birds at the Estaci\u00f3n de Biolog\u00eda Los Tuxtlas (from Howell and Webb, 1995). Habitat Foraging Elevational Geographic Species preference guild distribution (m) distribution Phaethornis striilgularis forest nectarivore 0-1500 periphery forest frugivore 0-1500 periphery forest frugivore 0-2400 core forest insectivore 0-1000 periphery forest insectivore 0-1500 core forest insectivore 0-1200 periphery semi-open insectivore 0-1500 periphery forest insectivore 0-1600 periphery forest insectivore 0-750 periphery forest insectivore 50-3500 core edge insectivore 0-1500 periphery edge insectivore 0-1300 periphery forest insectivore 0-1200 periphery forest insectivore 0-1000 periphery forest frugivore 0-750 periphery forest insectivore 0-1300 core edge frugivore 0-1000 periphery forest frugivore 0-750 periphery Hylomanes momotula Trogon collaris Xenops minutus Xiphorhynchus flavigaster Glyphorynchus spirurus* Lepidocolaptes souleyetii Taraba major* Formicarius analis* Grallaria guatimalensis* Ornithion semiflavum Leptopogon amaurocephalus Onychorhynchus coronatus Eucometis penicillata Myiobius sulphureipygius Schiffornis turdina* Henicorhina leucosticta Coereba flaveola PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t * Presence/Absence data suggest species is extirpated. PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t g guatimalensis, l. amaurocephalus, m. sulphureipygius, s. turdina, h. : leucosticta, and E. penicillata. L. souleyetii prefers semi-open or partly cleared forest. Eleven of 16, or 68.8%, of the species showing declines or extirpations in this study are insectivores, whereas among all species captured 41% are insectivores. This trend was not significant, however (G-test with Williams\u2019 correction, P > 0.1). The Sierra de Los Tuxtlas is the northernmost limit of the ranges of 13 of the 16 species showing declines. G. guatimalensis and H. leucosticta are the only species with a distribution extending substantially to the north and west of the study site. The field site is well within the elevational limits for all 16 species (Table 4). The two species that significantly increased in abundance over the sample period (Table 4) both occur here at the core of their ranges, elevational distributions, and in their preferred forest habitat. T. collaris is a frugivore, and X. flavigaster is an insectivore. DISCUSSION Although the absence of a species is not a clear indication of extirpation, our sampling effort, despite its heterogeneity, does suggest that at minimum a species\u2019 absence indicates a decline. It is possible that some of the species now apparently gone from the station may persist in other, unsampled fragments. If the data presented here and our interpretation of them are accurate, the extirpation of species from the Estaci\u00f3n de Biolog\u00eda Los Tuxtlas has been ongoing since its isolation. Such an \u201cextinction debt\u201d is 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t a recognized component of deforestation, and models of empirical data show that in birds this occurs across decades, but the species affected and the mechanisms of species loss remain poorly understood (Tilman et al., 1994; Ewers & Didham, 2003, Robinson & Sherry, 2012). Since 1973, 16 species susceptible to capture in mist nets have either become locally extirpated or are showing significant declines in abundance. The total number of losses and declines is undoubtedly higher than presented, because species not regularly captured in mist nets, such as large-bodied and canopy species, were not adequately surveyed. Species known to have been extirpated from Los Tuxtlas include Sarcoramphus papa, Harpia harpyja, and Ara macao. Patten, G\u00f3mez de Silva, & Smith-Patten (2010) also documented the extirpation of the latter two in Chiapas, Mexico. Many additional species have also been categorized as endangered or threatened in the Sierra de Los Tuxtlas (see Winker, 1997). Our estimate of the average rate of avian losses from the station of 3.7 species per decade may not be directly comparable to other studies due to differences in habitat and sampling, but it is similar to the rate of loss observed at Barro Colorado Island by Robinson (1999) of 3.3 species per decade. Our estimate, however, includes only those taxa captured in mist nets, whereas Robinson\u2019s work included all species detected through observation. Of the eight species with data sufficient for statistical analysis that showed local extirpation, six were lost between 1992 and 2004 (on the same site), suggesting a continuing extirpation of species from the station. 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t Bierregaard & Lovejoy (1988, 1989) found that as surrounding habitat was lost, species richness in remaining fragments increased as individuals displaced from surrounding areas found their way to remaining forest patches. This increased richness was limited by the lifespan of the individual birds (Bierregaard & Lovejoy 1988, 1989). Unlike these studies, in which forest patches were suddenly and completely isolated, the forest of the Estaci\u00f3n de Biolog\u00eda Los Tuxtlas was isolated gradually. Because extirpation seems to be continuing, we expect declines and extinctions to continue for some time at the station, even if no further deforestation occurs in the region (Willis, 1974; Brooks, Pimm, & Oyugi, 1999, Robinson, 1999; Ferraz et al., 2003). Mechanisms for tropical bird species losses due to deforestation and fragmentation probably include factors such as greater specialization as compared to temperate birds, reduced dispersal abilities, lower population densities, and patchy distributions (Robinson et al., 2004; Stratford & Robinson, 2005; Moore et al., 2008; Rompr\u00e9 et al., 2007). Our assessment of possible causes for the loss of these species reveals no definite patterns, however, other than the predominant requirement of forested habitat. On Barro Colorado Island in Lake Gat\u00fan, Panama, maturation of habitat and loss of open areas was responsible for the decline in the island\u2019s avifauna (Willis, 1974; Karr, 1982). This is unlikely to be the case in Los Tuxtlas. Despite major degradation of surrounding forests, the station has remained primary forest with areas of second growth. A loss of sapling and seedling species has been described (Dirzo & Miranda, 1990), but the overall structure of the forest 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t appears to have remained fairly stable. Vetter et al. (2011) found in a meta-analysis of 30 studies that the effects of fragmentation are not subject to simple generalities, and that they are highly site specific. Los Tuxtlas is at the northernmost extent of the ranges of 13 of the 16 species we found to be declining or extirpated (Tables 3 and 4). Evidence is mixed as to whether populations at the periphery of a species\u2019 range are more vulnerable to extinction (Terborgh & Winter, 1980; Kattan, Halvarez-Lopez, & Giraldo, 1994; Johnson, 1998). Los Tuxtlas is at the edge of all species\u2019 geographic ranges endemic to Neotropical rainforest, so it is not clear why this subset might be more subject to this phenomenon. The elevational distribution of each of these species encompasses sea level to 750 m or more (Howell & Webb, 1995), and we consider this factor unlikely to be responsible for the vulnerability of these particular taxa. Although insectivores showed a trend toward being disproportionately affected in our study, it was not significant. Elsewhere insectivores have been shown to be particularly vulnerable to severe habitat change (e.g., Kattan, Halvarez-Lopez, & Giraldo, 1994; Canaday, 1996; Johnson & Winker, 2010; Vetter et al., 2011). Additionally, deforestation can negatively impact species found in multi-species foraging flocks (Van Houtan et al., 2006), which are important to many birds of tropical rainforest communities (Willis, 1966; Morton, 1973; Buskirk, 1976; Rappole et al., 1983). Rappole & Morton (1985) noted that X. minutus, one of the species showing a significant decline in our study (Table 2) was a regular member of mixed flocks in the Sierra de Los Tuxtlas. 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t We considered large-scale range shifts, perhaps from climate change, as a possible cause for species loss, but this seems unlikely. At least some of the species lost in our study appear to have persisted in the southern portion of Los Tuxtlas near Volc\u00e1n Santa Marta at least into the mid-1990s (Winker, pers. obs.). If range shifts were the cause, species would likely have disappeared region-wide and we would not expect only forest-related species to be affected. Habitat loss and degradation seem to be the best explanations for the losses observed, but exactly how these changes affected each species remains unknown. Another possible influence on mist-net captures, particularly in the most recent, late winter/spring sampling periods, would be intra-tropical and elevational movements in some of the study species (Ramos, 1983, 1988). There is evidence that C. flaveola and E. flavogaster move seasonally within the tropics, seemingly to breed in Los Tuxtlas then departing (Ramos & Rappole, pers. obs.). Vega Rivera (1982) found probable elevational movements in M. sulphureipygius. The local extinctions of seven of the 16 species are particularly notable. C. flaveola is a widely distributed species known to thrive in manipulated habitats such as gardens and forest edges and is a generalist frugivore and nectarivore (pers. obs.; Howell & Webb, 1995). This is not a species we would expect to decline due to forest fragmentation; both its habitat and food preferences are well suited to survival in a mosaic landscape, and it is known to persist in a fragmented landscape elsewhere in northern Middle America (Johnson & Winker, 2010). Intratropical migrations of C. flaveola may partially explain the changing 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t capture rates in this species (Ramos & Rappole, pers. obs.). O. semiflavum and L. amaurocephalus are both edge specialists; thus, limited fragmentation, creating an increase in edges, might a priori seem to benefit these species. Though the habitat protected by the station has remained relatively static, the intensity of lowland deforestation in Los Tuxtlas as a whole (Fig. 2) may be too extensive even for these edge specialists. L. souleyetii prefers open forest and partially cleared areas (Howell & Webb, 1995). The habitat surrounding the station during the 1980s and 1990s was dominated by pasture scattered with isolated trees. In our later field seasons there was a noticeable decline in the number of isolated trees and fences constructed of living trees (Winker, pers. obs.). This loss may account for the extirpation of L. souleyetii. G. spirurus apparently disappeared from the station between the 1970s and 1986, the first of the documented extirpations. The majority of deforestation across the region took place during this period. This previously abundant species disappeared from our data in just over a decade. Interestingly, on the slopes of neighboring Volc\u00e1n Santa Marta the species was present at least into the 1990s and probably still persists there (Winker, pers. obs.). Also, Estrada, Coates-Estrada, & Merritt (1997) had observational data of the species\u2019 presence in the station area in 1990-1992, indicating at least a decline if not extirpation (Table 2). In Brazil, G. spirurus persisted in experimentally isolated fragments well after isolation (Stouffer & Bierregaard 1995), and the species persists in highly fragmented forest in southern Belize (Johnson & Winker, 2010). H. momotula was collected but not netted in 1974, was captured in substantial numbers during 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t 1986 and 1992-94, but was absent in the last two seasons of sampling. This pattern is mysterious. This species has an elevational range extending to 1500 m and may persist in the forests of the upper slopes of Volc\u00e1n San Mart\u00edn. If so, we speculate that the station may serve as a sink for this species, where habitat is insufficient for a self-sustaining population but may occasionally be colonized by dispersing individuals (see also Winker et al., 1997). Continued sampling may provide more insight into its abundance patterns. It illustrates the need for improved understanding of species-specific dispersal behavior within and among forest fragments (e.g., Van Houtan et al., 2007; Moore et al., 2008; Ibarra-Macias, Robinson, & Gaines 2011), which may be an important driver for patterns such as those we observed. Two other studies provide comparative value to our results. The four species we consider likely extirpated (Taraba major, Formicarius analis, Grallaria guatimalensis, and Schiffornis turdina) were not detected in the much broader census surveys of Estrada, Coates-Estrada, & Merritt (1997) in 1990-1992. Patten, G\u00f3mez de Silva, & Smith-Patten (2010) conducted the geographically closest long-term study to ours in their analysis of avian declines at Palenque, Chiapas, Mexico. Their results showed only three species that overlapped our results. They found Eucometis penicillata extirpated (to our decline) and two others that declined as our populations did (Xenops minutus and Leptopogon amaurocephalus). Indeed, the species-level heterogeneity between our studies is noteworthy. Our analyses suggest that the Estaci\u00f3n de Biolog\u00eda Tropical Los Tuxtlas 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t is too small to maintain its full, historic complement of bird species. If deforestation accelerated region-wide, eliminating other forest refugia, the station alone (640 ha) would be unable to maintain the historical avian diversity of the region or to provide source populations for restored forest habitats for many of its present bird species. Given the scale of deforestation in the region, it is surprising that there are not more species showing declines. Indeed, we may consider it good news that important forest seed dispersers such as Habia tanagers (Puebla & Winker, 2004) did not show significant declines. The overall size of the remaining forests, particularly in the highlands, may be ameliorating the effects of lowland deforestation. However, increasing or continued isolation of the station will probably limit recolonization from elsewhere, and species losses will likely continue. In our study, although several species seemed to quickly succumb to local and regional deforestation, others showed delayed declines and extirpations, a phenomenon also known to happen at larger scales (Tilman et al., 1994, Pimm et al., 2006). Moreover, the effects of deforestation were remarkably heterogeneous among forest-related species, with no single clear pattern of why some species experienced declines or extirpation. Our long-term data suggest that predicting which species will be most affected by deforestation in the northern Neotropics, and thus effectively working to ameliorate the effects of forest loss, will be particularly challenging. Nevertheless, as similar long-term datasets accrue, subtle patterns may reveal how species-specific responses reflect underlying commonalities that can be exploited for effective management and conservation. 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t acknowledgments : We thank the many field assistants who have helped us over the years and those who have provided and helped obtain permits to conduct this research. R. Barry, A. Powell, S. Pimm, and three anonymous reviewers provided excellent advice and comments. LITERATURE CITED Andr\u00e9n H. 1994. Effects of habitat fragmentation on birds and mammals in landscapes with different proportions of suitable habitat: a review. Oikos 71:355-366. Andrle RF. 1966. North American migrants in the Sierra de Tuxtla of southern Veracruz, Mexico. Condor 68:177-184. Barlow J, Peres CA, Henriques LMP, Stouffer PC, Wunderle JM. 2006. The responses of understorey birds to forest fragmentation, logging and wildfires: An Amazonian synthesis. Biological Conservation 128:182-192. Becker P, Moure JS, Peralta FJA. 1991. More about euglossine bees in Amazonian forest fragments. Biotropica 23:586-591. Bierregaard RO, Lovejoy TE. 1988. Birds in Amazonian forest fragments: Effects of insularization. Pages 1564-1579 in: Acta XIX Congressus Internationalis Ornithologici (H. Oellet, ed.). University of Ottawa Press, Ottawa, Canada. Bierregaard RO, Lovejoy TE. 1989. Effects of forest fragmentation on Amazonian understory bird communities. Acta Amazonica 19:215-241. Blake JG. 1991. Nested subsets and the distribution of birds in isolated 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t woodlots. Conservation Biology 5:58-86. Brook BW, Sodhi NS, Ng PKL. 2003. Catastrophic extinctions follow deforestation in Singapore. Nature 424:420-423. Brooks TM, Pimm SL, Oyugi JO. 1999. Time lag between deforestation and bird extinction in tropical forest fragments. Conservation Biology 13:1140-1150. Buskirk WH. 1976. Social systems in a tropical forest avifauna. American Naturalist 110:293-310. Canaday C. 1996. Loss of insectivorous birds among a gradient of human impact in Amazonia. Biological Conservation 77:63-77. Ceballos G, Ehrlich PR. 2002. Mammal population losses and the extinction crisis. Science 296:904-907. Daily GC, Ehrlich PR. 1995. Preservation of biodiversity in small rainforest patches: rapid evaluations using butterfly trapping. Biodiversity and Conservation 4: 35-55. Dirzo R, Garcia MC. 1992. Rates of deforestation in Los Tuxtlas, a Neotropical area in southeast Mexico. Conservation Biology 6:84-90. Dirzo R, Miranda A. 1990. Contemporary Neotropical defaunation and forest structure, function, and diversity, a sequel to John Terborgh. Conservation Biology 4:444-447. Dirzo R, Miranda A. 1991. Altered patterns of herbivory and diversity in the forest understory: a case study of the possible consequences of contemporary defaunation. Pages 273-287 in: Plant-Animal Interactions: Evolutionary ecology in tropical and temperate regions 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t (Price PW, Lewinsohn TM, Fernandes GW, Benson WW, eds.). Wiley and Sons, Inc., New York. Dirzo R, Raven PH. 2003. Global state of biodiversity and loss. Annual Review of Environment and Resources 28:137-167. Estrada A, Coates-Estrada R, Merritt Jr DA. 1997. Anthropogenic landscape changes and avian diversity at Los Tuxtlas, Mexico. Biodiversity and Conservation 6:19-43. Ewers RM, Didham RK. 2006. Confounding factors in the detection of species responses to fragmentation. Biological Reviews 81:117-142. Faaborg J, Brittingham M, Donovan T, Blake J. 1995. Habitat fragmentation in the temperate zone. Pages 357-380 in: Ecology and management of Neotropical migratory birds: a synthesis and review of the critical issues (Finch DM, Martin TE, eds.). Oxford University Press, Cambridge, United Kingdom. Fahrig L. 2003. Effects of habitat fragmentation on biodiversity. Annual Review of Ecology Evolution and Systematics 34:487-515. Ferraz G, Russell GJ, Stouffer PC, Bierregaard RO, Pimm SL, Lovejoy TE. 2003. Rates of species loss from Amazonian forest fragments. Proceedings of the National Academy of Sciences USA 100:14069-14073. Ferraz G, Nichols JD, Hines JE, Stouffer PC, Bierregaard RO, Lovejoy TE. 2007. A large-scale deforestation experiment: Effects of patch area and isolation on Amazon birds. Science 315:238-241. Gonz\u00e1lez-Soriano E, Dirzo R, Vogt RC, eds. 1997. Historia Natural de Los Tuxtlas. Universidad Nacional Aut\u00f3noma de M\u00e9xico, M\u00e9xico, D.F. 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t Howell S, Webb S. 1995. A guide to the birds of Mexico and northern Central America. Oxford University Press, New York. Ibarra-Macias A, Robinson WD, Gaines MS. 2011. Experimental evaluation of bird movements in a fragmented Neotropical landscape. Biological Conservation 144:703-712. Ibarra-Manr\u00edquez G, Martinez-Ramos M, Dirzon R, N\u00fa\u00f1ez-Farf\u00e1n J. 1997 La vegetaci\u00f3n. Pages 61\u201374 in Historia Natural de Los Tuxtlas (Gonz\u00e1lez-Soriano E, Dirzo R, Vogt RC, Eds). Universidad Nacional Aut\u00f3noma de M\u00e9xico, M\u00e9xico, D.F. Johnson AB, Winker K. 2010. Short-term hurricane impacts on a Neotropical community of marked birds. PLoS ONE 5:e15109. Johnson CN. 1998. Species extinction and the relationship between distribution and abundance. Nature 394:272-274. Kattan GH, Halvarez-Lopez H, Giraldo M. 1994. Forest fragmentation and bird extinctions: San Antonio eighty years later. Conservation Biology 8:138-146. Karr JR. 1982. Population variability and extinction in a tropical land-bridge island. Ecology 63:1975-1978. Laurance WF, et al. 2011. The fate of Amazonian forest fragments: A 32-year investigation. Biological Conservation 144:56-67. Leck CF. 1979. Avian extinctions in an isolated tropical wet-forest preserve, Ecuador. Auk 96:343-352. Lees AC, Peres CA. 2006. Rapid avifaunal collapse along the Amazonian 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t deforestation frontier. Biological Conservation 133:198-211. Leigh EG, Jr. 1975. Population fluctuations, community stability, and environmental variability. Pages 52-73 in: Ecology and evolution of communities (Cody ML, Diamond JM, eds.). Belknap Press, Cambridge, Massachusetts. Leigh EG, Jr. 1981. The average lifetime of a population in a varying environment. Journal of Theoretical Biology 90:213-239. Lovejoy TE, Rankin JM, Bierregaard RO, Brown KS, Jr., Emmons LH, Van Der Voort ME. 1984. Ecosystem decay of Amazon forest remnants. Pages 295-325 in: Extinctions (Nitecki MH, ed.). University of Chicago Press, Chicago, Illinois. Lovejoy TE, Bierregaard RO, Rylands AB, Malcolm JR, Quintela CE, Harper LH, Brown KS, Powell AH, Powell GVN, Schubart HOR, Hays MB. 1986. Edge and other effects of isolation on Amazon forest fragments. Pages 257-285 in: Conservation biology: The science of scarcity of diversity (Soule ME, ed.). Sinauer Associates, Sunderland, Massachusetts. Malcolm JR. 1988. Small mammal abundances in isolated and non-isolated primary forest reserves near Manaus, Brazil. Acta Amazonica 18: 67-83. Mendoza E, Fay J, Dirzo R. 2005. A quantitative analysis of forest fragmentation in Los Tuxtlas, southeast Mexico: patterns and implications for conservation. Revista Chilena de Historia Natural 78:451-467. Moore RP, Robinson WD, Lovette IJ, Robinson TR. 2008. Experimental evidence for extreme dispersal limitation in tropical forest birds. 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t Ecology Letters 11: 960-968. Morton ES. 1973. On the evolutionary advantages and disadvantages of fruit eating in tropical birds. American Naturalist 107:8-22. Nemark WD. 1991. Tropical forest fragmentation and the local extinction of understory birds in the eastern Usambara Mountains, Tanzania. Conservation Biology 5:67-78. O\u2019Grady JJ, Reed DH, Brook BW, Frankham R. 2004. What are the best correlates of predicted extinction risk? Biological Conservation 118:513-520. Pahl LI, Winter JW, Heinsohn G. 1988. Variation in responses of arboreal marsupials to fragmentation of tropical rainforest in north eastern Australia. Biological Conservation 46:71-82 Patten MA, Gomez de Silva H, Smith-Patten BD. 2010. Long-term changes in the bird community of Palenque, Chiapas, in response to rainforest loss. Biodiversity and Conservation 19:21-36. Pennington TD, Sarukhan J. 1968. Arboles Tropicales de Mexico. Instituto Nacional de Investigacciones Forestales, D.F., Mexico. Pimm SL, Jones HH, Diamond J. 1988. On the risk of extinction. American Naturalist 132:757-785. Pimm SL, Raven P, Peterson A, \u015eekercio\u011flu \u00c7, Ehrlich PR. 2006. Human impacts on the rates of recent, present, and future bird extinctions. Proceedings of the National Academy of Sciences USA 103:10941-10946. Powell AH, Powell GVN. 1987. Population dynamics of male euglossine bees in 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t Amazonian forest fragments. Biotropica 19: 176-179. Puebla F, Winker K. 2004. Dieta y dispersi\u00f3n de semillas de dos especies de tangara (Habia) en dos tipos de vegetaci\u00f3n en Los Tuxtlas, Veracruz, M\u00e9xico. Ornitolog\u00eda Neotropical 15:53-64. Ramos MA. 1983. Seasonal movements of bird populations at a Neotropical study site in southern Veracruz, Mexico. Ph.D. dissertation. University of Minnesota, Minneapolis. Ramos MA. 1988. Eco-evolutionary aspects of bird movements in the northern Neotropical region. Pages 251-293 in: Acta XIX Congressus Internationalis Ornithologici, Vol. I, (Ouellet H, ed.). University of Ottawa Press, Ottawa, Canada. Rappole JH, Warner DW. 1980. Ecological aspects of migrant bird behavior in Veracruz, Mexcio. Pages 353-393 in: Migrant Birds in the Neotropics: Ecology, Behavior, Distribution, and Conservation, (Keast A, Morton ES, eds.). Smithsonian Institution Press, Washington, D. C. Rappole JH, Morton ES, Lovejoy TE, III, Ruos JL. 1983. Nearctic avian migrants in the Neotropics. United States Fish and Wildlife Service, Washington D.C. Rappole JH, Powell GVN, Sader SA. 1994. Remote-sensing assessment of tropical habitat availability for a Nearctic migrant: the Wood Thrush. Pages 91-103 in: Mapping the diversity of nature (Miller RI, ed.). Chapman and Hall, London. Rappole JH, Winker K, Powell GVN. 1998. Migratory bird habitat use in southern Mexico: mist nets versus point counts. Journal of Field 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t Ornithology 69:635-643. Remsen JV Jr., Good DA. 1996. Misuse of data from mist-net captures to assess relative abundance in bird populations. Auk 113: 381-398. Robbins CS. 1980. Effect of forest fragmentation on breeding bird populations in the piedmont of the mid-Atlantic region. Atlantic Naturalist 33:31-36. Robinson WD. 1999. Long-term changes in the avifauna of Barro Colorado Island, Panama, a tropical forest isolate. Conservation Biology 13:85-97. Robinson WD, Sherry TW. 2012. Mechanisms of avian population decline and species loss in tropical forest fragments. Journal of Ornithology 153:S141-S152. Robinson WD, Angehr GR, Robinson TR, Petit LJ, Petit DR, Brawn JD. 2004. Distribution of bird diversity in a vulnerable Neotropical landscape. Conservation Biology 18:510-518. Rolstad J. 1991. Consequences of forest fragmentation for the dynamics of bird populations: conceptual issues and the evidence. Biological Journal of the Linnean Society 42:149-163. Rompr\u00e9 G, Robinson WD, Desrochers A, Angehr G. 2007. Environmental correlates of avian diversity in lowland Panama rainforests. Journal of Biogeography 34:802-815. Schaldach W, Escalante P. 1997. Lista de Aves. Pages 571-573 in: Historia Natural de Los Tuxtlas (Gonz\u00e1lez-Soriano E, Dirzo R, Vogt RC, eds.). Universidad Nacional Autonoma de Mexico. D.F., Mexico. Sodhi NS, Liow LH, Bazzaz FA. 2004. Avian extinctions from tropical and subtropical forests. Annual Review of Ecology Evolution and 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t Systematics 35:323-345. Soto M, Gama L. 1997. Climas. Pages 7-23 In: Historia Natural de Los Tuxtlas (Gonz\u00e1lez-Soriano E, Dirzo R, Vogt RC, eds.). Universidad Nacional Autonoma de Mexico. D.F., Mexico. Stotz DF, Fitzpatrick JW, Parker TA, Moskovits DK. 1996. Neotropical birds: ecology and conservation. University of Chicago Press, Chicago. Stouffer PC, Bierregaard RO. 1995. Effects of forest fragmentation on understory insectivorous birds. Ecology 76:2429-45. Stratford JA, Robinson WD. 2005. Gulliver travels to the fragmented tropics: geographic variation in mechanisms of avian extinction. Frontiers in Ecology and the Environment 3:85-92. Stuart SN, Chanson JS, Cox NA, Young BE, Rodrigues ASL, Fischman DL, Waller RW. 2004. Status and trends of amphibian declines and extinctions worldwide. Science 306:1783-1786. Terborgh J, Winter B. 1980. Some causes of extinction. Pages 119-133 In: Conservation biology: an evolutionary-ecological perspective (Soule ME, Wilcox BA, eds.). Sinauer Associates, Sunderland, Massachusetts. Tilman D, May RM, Lehman CL, Nowak MA. 1994. Habitat destruction and the extinction debt. Nature 371:65-66. Turner IM. 1996. Species loss in fragments of tropical rain forest: a review of the evidence. Journal of Applied Ecology 33:200-209. Van Houtan KS, Pimm SL, Bierregaard RO, Jr., Lovejoy TE, Stouffer PC. 2006. Local extinctions in flocking birds in Amazonian forest fragments. Evolutionary Ecology Research 8:129-148. 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t Van Houtan KS, Pimm SL, Halley JM, Bierregaard RO Jr., Lovejoy TE. 2007. Dispersal of Amazonian birds in continuous and fragmented forest. Ecology Letters 10:219-229. Vega Rivera JH. 1982. Aspectos biologicos de Myiobius sulphureipygius (Aves: Tyrannidae) en el area de Santa Marta region de \u201cLos Tuxtlas,\u201d Veracruz, Mexico. Professional thesis. Universidad Nacional Autonoma de Mexico. Vetter D, Hansbauer MM, V\u00e9gv\u00e1ri Z, Storch I. 2011. Predictors of forest fragmentation sensitivity in Neotropical vertebrates: a quantitative review. Ecography 34:1-8. Whitman AA, Hagan JM III, Brokaw NVL. 1997. A comparison of two survey techniques used in a subtropical forest. Condor 99:955-965. Willis EO. 1966. The role of migrant birds at swarms of army ants. Living Bird 5:187-231. Willis EO. 1974. Populations and local extinctions of birds on Barro Colorado Island, Panama. Ecological Monographs 44:153-169. Willis EO. 1979. The composition of avian communities in remanescent woodlots in southern Brazil. Papeis Avulsos de Zoolgia 33:1-25. Winker K. 1995. Habitat selection in woodland Nearctic-Neotropic migrants on the Isthmus of Tehuantepec. I. Autumn migration. Wilson Bulletin 107:26-39. Winker K. 1997. Introduction to the birds of Los Tuxtlas. Pages 535-541 in: Historia Natural de Los Tuxtlas (Gonz\u00e1lez-Soriano E, Dirzo R, Vogt RC, Eds.). Universidad Nacional Autonoma de Mexico. D.F., Mexico. 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t Winker K, Rappole JH, Ramos MA. 1990. Population dynamics of the Wood Thrush (Hylocichla mustelina) on its wintering grounds in southern Veracruz, Mexico. Condor 92:444-460. Winker K, Oehlenschlager RJ, Ramos MA, Zink RM, Rappole JH, Warner DW. 1992. Avian distribution and abundance records for the Sierra de Los Tuxtlas, Veracruz, Mexico. Wilson Bulletin 104:699-718. Winker K, Escalante P, Rappole JH, Ramos MA, Oehlenschlager RJ, Warner DW. 1997. Periodic migration and lowland forest refugia in a sedentary Neotropical bird, Wetmore\u2019s bush-tanager. Conservation Biology 11:692-697. Zimmerman BL, Bierregaard RO. 1986. Relevance of the equilibrium theory of island biogeography and species-area relations to conservation with a case from Amazonia. Journal of Biogeography 13:133-143. 702 703 704 705 706 707 708 709 710 711 712 713 714 PeerJ reviewing PDF | (v2013:07:699:0:1:NEW 1 Aug 2013) R ev ie w in g M an us cr ip t",
    "url": "https://peerj.com/articles/180/reviews/",
    "review_1": "Silvia Moreno \u00b7 Sep 18, 2013 \u00b7 Academic Editor\nACCEPT\nThe author response to the critique is appropriate and the manuscript is acceptable for publication in PeerJ.",
    "review_2": "Silvia Moreno \u00b7 Dec 29, 2012 \u00b7 Academic Editor\nMAJOR REVISIONS\nYour manuscript was reviewed by two experts and both reviewers have concerns about the presented data and request additional experiments/controls. I would re-consider a re-submission but it is critical that the major concerns identified in the critique be dealt with appropriately.",
    "review_3": "Reviewer 1 \u00b7 Dec 28, 2012\nBasic reporting\nSee below.\nExperimental design\nSee below.\nValidity of the findings\nSee below.\nAdditional comments\nIn this study, Klein et al. proposes that T. brucei SUMO modifies proteins and is critical for growth of bloodstream forms. The authors also suggest that SUMOylation is elevated under oxidative stress but is unchanged by temperature variation. Finally, the authors have identified two gene homologues that likely encode the E2 SUMO-conjugating enzyme (Ubc9) and the SUMO-specific protease (SENP). The paper is somewhat interesting (especially regarding the effect of oxidative stress on SUMOylation) but, disappointingly, it falls short in some key originality and experimental/methodological aspects (see below). Moreover, most of the results seem very preliminary at this point and additional experiments should be carried out, particularly concerning detection, identification, and validation of the potential SUMOylated proteins, and the role of SUMOylation in oxidative stress.\n\nMajor issues:\n\n1) SUMOylation has recently been described in T. brucei bloodstream forms by Obaldo et al. (Nucleic Acids Res, 39:1023-33, 2011). Several of the observations regarding the role of SUMOylation in parasite cell growth currently described in this manuscript have already been reported by Obado et al. Thus, there is no major novelty on the data presented here. By the way, that key reference is surprisingly absent from the manuscript.\n\n2) Failure in purifying and identifying any bloodstream SUMOylated protein using tandem-affinity purification (TAP) or V-5 tag. On this regard, there are many technical issues that could result in a negative TAP or V-5 purification outcome, some of them rightly pointed out by the authors: insufficient amount of protein in the initial cell lysate, low specificity of the immobilized antibody, inappropriate inhibition of SUMO proteases, low sensitivity of the methodology used for detecting the SUMOylated proteins. For instance, the authors have used SDS-PAGE trying to detect proteins after elution by TEV cleavage. It is unclear, however, what kind of gel staining they employed. This could make a huge difference in terms of sensitivity. Furthermore, very hydrophobic, high molecular mass, heavily posttranslationally modified, and/or of low abundance proteins that are not detected by SDS-PAGE (even using highly sensitive silver or fluorescence-based staining) could be identified by more sensitive approaches, such as in-solution trypsin digestion followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Unfortunately, the authors did not explore this possibility. Also, it is unclear why the majority of TAP-tagged SUMOylated proteins could not be eluted from IgG beads. There is no discussion in that regard.\n\nMinor issues:\n\n1) Page 5, lines 1-2, and throughout the text, numbers with decimals are not in English format (e.g., 62,5 uM)\n\n2) V5 Immunoprecipitation: What type of IGEPAL detergent was used? CA-630 (octylphenoxy poly(ethyleneoxy)ethanol)? There are over 30 different types of IGEPAL.\n\n3) Please specify how exactly anti-V5 antibodies were coupled to the column? Was it by the cyanogen bromide method? The reference by Harlow and Lane (1999) is a handbook with many different coupling methods.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"SUMOylation in Trypanosoma brucei (v0.1)\". PeerJ https://doi.org/10.7287/peerj.180v0.1/reviews/1",
    "review_4": "Reviewer 2 \u00b7 Dec 19, 2012\nBasic reporting\nAccording to the abstract, the authors say that they demonstrate:\n1) that SUMO modifies several proteins in T. brucei; 2) that the SUMOylation levels are not affected by changes in temperature, but are increased by oxidative stress; 3) that SUMO is required for growth of the bloodstream forms; and 4) that they obtained evidence indicating that the E2 and SENP homologs are involved in SUMOylation and the removal of SUMO, respectively.\nAbout points 1 and 2:\nThat SUMO modifies a number of proteins in T. brucei has already been demonstrated by Obado et al (ref. 1 ) for the bloodstream forms.\nThe authors do not detect the endogenous SUMOylation pattern because of the lack of specificity of their antibodies, generated against the purified recombinant protein TbSUMO. In order to detect a SUMOylation pattern they had to do a partial replacement (of only one allele) of the endogenous gene by an N-terminal tagged form. In the case of the procyclics they use the V5 epitope and in the case of bloodstream forms the TAP tag, and then they use the corresponding commercial antibodies. The problem is, particularly when they use the 20 kDa TAP tag, that probably the tagged SUMO may not be conjugated to target proteins as efficiently as endogenous SUMO. Thus, the SUMOylation pattern presented both for normal growth conditions or under different stress conditions might not be representative of what is really occurring in the wild type parasite.\nThe authors do not see changes in the SUMOylation pattern either during thermal stress, or during differentiation, but they see changes during oxidative stress of the procyclic forms. However, as the authors themselves comment, in these experiments only the more abundant proteins are detected, which may not necessarily change under stress conditions; they can not discard the possibility that less abundant proteins have modified SUMOylation levels, but are not detected.\nAbout point 3:\nThis has already been done and published before. The authors do not make reference to the SUMO RNAi experiments performed by Obado et al with bloodstream parasites: \"The SUMO transcript was almost completely depleted within 24 h of RNAi induction (Figure 5B). However, it took at least 48 h until there was an effect on the global sumoylation pattern (Figure 5C). Western analysis of parasite lysates identified a range of sumoylated proteins, with varying intensity. The parasites were found to stop dividing after 24 h of SUMO RNAi, significant cell death was not observed until beyond the 96-h time point, nuclear division appeared to continue in the absence of cytokinesis, with many cells displaying a multi-nucleated phenotype.\"\nAbout point 4) The orthologs of E2 and SENP from T. brucei were identified in silico. The authors do not demonstrate the biochemical activity of these proteins, but instead they study indirectly how the downregulation of their levels affects the global SUMOylation pattern in procyclics. The RNAi experiments are very preliminary. The authors do not show how the levels of RNA (using Northern Blot of qPCR) and protein (by Western Blot) are affected. The growth curves are not shown, and different clones are not analyzed.\nIn summary, this manuscript has a number of flaws in experimental design, and part of the results has little novelty, suggesting that its publication in its present form would be premature.\nRef 1:\nObado SO, Bot C, Echeverry MC, Bayona JC, Alvarez VE, Taylor MC, Kelly JM.\nCentromere-associated topoisomerase activity in bloodstream form Trypanosoma brucei.\nNucleic Acids Res. 2011 Feb;39(3):1023-33\nExperimental design\nAbout the section on \"Failure to purify SUMOylated proteins from T. brucei extracts\":\nIn the case of the bloodstream forms, the reason for this failure can be a combination of factors:\n- starting from a low number of parasites, as compared with the numbers of cells used for the purification of SUMOylated proteins from other organisms.\n- the utilization of a rather little efficient cell breakage method for the extraction of SUMOylated proteins (since it has already been demonstrated that they are essentially nuclear proteins in Trypanosomatids).\n- the insufficient inhibition of the deSUMOylases, because of the elimination of NEM during washing, or the use of a purification protocol under non-denaturing conditions.\nThe authors say:\n\"Eluates from cells expressing TAP-tagged SUMO gave exactly the same pattern on SDS-PAGE as eluates from cells expressing TAP alone and SUMO was not visible.\"\nWas this analyzed by SDS-PAGE, staining the gels with silver? with Coomassie? The pattern observed may be identical in these cases because the SUMOylated proteins normally represent a very minor fraction of each protein target. What happens when analysis is made by Western Blot? Do they see TAP-SUMO? If they do, this would mean that most of the SUMOylated proteins were deconjugated during purification; if they do not see TAP-SUMO, this means that elution has failed.\n\"Further analysis indicated that the majority of TAP-tagged SUMO had bound to the IgG beads, but it was never recovered. Additional bands were however not seen after analysis of the boiled beads.\"\nAccording to this statement, elution is what is not working in these experiments.\nWhen purification from procyclics was attempted by immunoaffinity, clearly this did not work, but this does not mean that the SUMOylated proteins can not be purified.\nSummarizing, the Results section entitled \"Failure to purify SUMOylated proteins from T. brucei extracts\" is too long and confusing. I suggest to eliminate or substantially shorten this section, saying, as Annoura et al did, that \"failure of the detection of SUMOylated proteins after purification might be due to the rapid turnover of SUMO conjugation.\"\nValidity of the findings\nThis manuscript has a number of flaws in experimental design, and part of the results has little novelty, suggesting that its publication in its present form would be premature.\nAdditional comments\nNo comments\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"SUMOylation in Trypanosoma brucei (v0.1)\". PeerJ https://doi.org/10.7287/peerj.180v0.1/reviews/2",
    "pdf_1": "https://peerj.com/articles/180v0.2/submission",
    "pdf_2": "https://peerj.com/articles/180v0.1/submission",
    "all_reviews": "Review 1: Silvia Moreno \u00b7 Sep 18, 2013 \u00b7 Academic Editor\nACCEPT\nThe author response to the critique is appropriate and the manuscript is acceptable for publication in PeerJ.\nReview 2: Silvia Moreno \u00b7 Dec 29, 2012 \u00b7 Academic Editor\nMAJOR REVISIONS\nYour manuscript was reviewed by two experts and both reviewers have concerns about the presented data and request additional experiments/controls. I would re-consider a re-submission but it is critical that the major concerns identified in the critique be dealt with appropriately.\nReview 3: Reviewer 1 \u00b7 Dec 28, 2012\nBasic reporting\nSee below.\nExperimental design\nSee below.\nValidity of the findings\nSee below.\nAdditional comments\nIn this study, Klein et al. proposes that T. brucei SUMO modifies proteins and is critical for growth of bloodstream forms. The authors also suggest that SUMOylation is elevated under oxidative stress but is unchanged by temperature variation. Finally, the authors have identified two gene homologues that likely encode the E2 SUMO-conjugating enzyme (Ubc9) and the SUMO-specific protease (SENP). The paper is somewhat interesting (especially regarding the effect of oxidative stress on SUMOylation) but, disappointingly, it falls short in some key originality and experimental/methodological aspects (see below). Moreover, most of the results seem very preliminary at this point and additional experiments should be carried out, particularly concerning detection, identification, and validation of the potential SUMOylated proteins, and the role of SUMOylation in oxidative stress.\n\nMajor issues:\n\n1) SUMOylation has recently been described in T. brucei bloodstream forms by Obaldo et al. (Nucleic Acids Res, 39:1023-33, 2011). Several of the observations regarding the role of SUMOylation in parasite cell growth currently described in this manuscript have already been reported by Obado et al. Thus, there is no major novelty on the data presented here. By the way, that key reference is surprisingly absent from the manuscript.\n\n2) Failure in purifying and identifying any bloodstream SUMOylated protein using tandem-affinity purification (TAP) or V-5 tag. On this regard, there are many technical issues that could result in a negative TAP or V-5 purification outcome, some of them rightly pointed out by the authors: insufficient amount of protein in the initial cell lysate, low specificity of the immobilized antibody, inappropriate inhibition of SUMO proteases, low sensitivity of the methodology used for detecting the SUMOylated proteins. For instance, the authors have used SDS-PAGE trying to detect proteins after elution by TEV cleavage. It is unclear, however, what kind of gel staining they employed. This could make a huge difference in terms of sensitivity. Furthermore, very hydrophobic, high molecular mass, heavily posttranslationally modified, and/or of low abundance proteins that are not detected by SDS-PAGE (even using highly sensitive silver or fluorescence-based staining) could be identified by more sensitive approaches, such as in-solution trypsin digestion followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Unfortunately, the authors did not explore this possibility. Also, it is unclear why the majority of TAP-tagged SUMOylated proteins could not be eluted from IgG beads. There is no discussion in that regard.\n\nMinor issues:\n\n1) Page 5, lines 1-2, and throughout the text, numbers with decimals are not in English format (e.g., 62,5 uM)\n\n2) V5 Immunoprecipitation: What type of IGEPAL detergent was used? CA-630 (octylphenoxy poly(ethyleneoxy)ethanol)? There are over 30 different types of IGEPAL.\n\n3) Please specify how exactly anti-V5 antibodies were coupled to the column? Was it by the cyanogen bromide method? The reference by Harlow and Lane (1999) is a handbook with many different coupling methods.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"SUMOylation in Trypanosoma brucei (v0.1)\". PeerJ https://doi.org/10.7287/peerj.180v0.1/reviews/1\nReview 4: Reviewer 2 \u00b7 Dec 19, 2012\nBasic reporting\nAccording to the abstract, the authors say that they demonstrate:\n1) that SUMO modifies several proteins in T. brucei; 2) that the SUMOylation levels are not affected by changes in temperature, but are increased by oxidative stress; 3) that SUMO is required for growth of the bloodstream forms; and 4) that they obtained evidence indicating that the E2 and SENP homologs are involved in SUMOylation and the removal of SUMO, respectively.\nAbout points 1 and 2:\nThat SUMO modifies a number of proteins in T. brucei has already been demonstrated by Obado et al (ref. 1 ) for the bloodstream forms.\nThe authors do not detect the endogenous SUMOylation pattern because of the lack of specificity of their antibodies, generated against the purified recombinant protein TbSUMO. In order to detect a SUMOylation pattern they had to do a partial replacement (of only one allele) of the endogenous gene by an N-terminal tagged form. In the case of the procyclics they use the V5 epitope and in the case of bloodstream forms the TAP tag, and then they use the corresponding commercial antibodies. The problem is, particularly when they use the 20 kDa TAP tag, that probably the tagged SUMO may not be conjugated to target proteins as efficiently as endogenous SUMO. Thus, the SUMOylation pattern presented both for normal growth conditions or under different stress conditions might not be representative of what is really occurring in the wild type parasite.\nThe authors do not see changes in the SUMOylation pattern either during thermal stress, or during differentiation, but they see changes during oxidative stress of the procyclic forms. However, as the authors themselves comment, in these experiments only the more abundant proteins are detected, which may not necessarily change under stress conditions; they can not discard the possibility that less abundant proteins have modified SUMOylation levels, but are not detected.\nAbout point 3:\nThis has already been done and published before. The authors do not make reference to the SUMO RNAi experiments performed by Obado et al with bloodstream parasites: \"The SUMO transcript was almost completely depleted within 24 h of RNAi induction (Figure 5B). However, it took at least 48 h until there was an effect on the global sumoylation pattern (Figure 5C). Western analysis of parasite lysates identified a range of sumoylated proteins, with varying intensity. The parasites were found to stop dividing after 24 h of SUMO RNAi, significant cell death was not observed until beyond the 96-h time point, nuclear division appeared to continue in the absence of cytokinesis, with many cells displaying a multi-nucleated phenotype.\"\nAbout point 4) The orthologs of E2 and SENP from T. brucei were identified in silico. The authors do not demonstrate the biochemical activity of these proteins, but instead they study indirectly how the downregulation of their levels affects the global SUMOylation pattern in procyclics. The RNAi experiments are very preliminary. The authors do not show how the levels of RNA (using Northern Blot of qPCR) and protein (by Western Blot) are affected. The growth curves are not shown, and different clones are not analyzed.\nIn summary, this manuscript has a number of flaws in experimental design, and part of the results has little novelty, suggesting that its publication in its present form would be premature.\nRef 1:\nObado SO, Bot C, Echeverry MC, Bayona JC, Alvarez VE, Taylor MC, Kelly JM.\nCentromere-associated topoisomerase activity in bloodstream form Trypanosoma brucei.\nNucleic Acids Res. 2011 Feb;39(3):1023-33\nExperimental design\nAbout the section on \"Failure to purify SUMOylated proteins from T. brucei extracts\":\nIn the case of the bloodstream forms, the reason for this failure can be a combination of factors:\n- starting from a low number of parasites, as compared with the numbers of cells used for the purification of SUMOylated proteins from other organisms.\n- the utilization of a rather little efficient cell breakage method for the extraction of SUMOylated proteins (since it has already been demonstrated that they are essentially nuclear proteins in Trypanosomatids).\n- the insufficient inhibition of the deSUMOylases, because of the elimination of NEM during washing, or the use of a purification protocol under non-denaturing conditions.\nThe authors say:\n\"Eluates from cells expressing TAP-tagged SUMO gave exactly the same pattern on SDS-PAGE as eluates from cells expressing TAP alone and SUMO was not visible.\"\nWas this analyzed by SDS-PAGE, staining the gels with silver? with Coomassie? The pattern observed may be identical in these cases because the SUMOylated proteins normally represent a very minor fraction of each protein target. What happens when analysis is made by Western Blot? Do they see TAP-SUMO? If they do, this would mean that most of the SUMOylated proteins were deconjugated during purification; if they do not see TAP-SUMO, this means that elution has failed.\n\"Further analysis indicated that the majority of TAP-tagged SUMO had bound to the IgG beads, but it was never recovered. Additional bands were however not seen after analysis of the boiled beads.\"\nAccording to this statement, elution is what is not working in these experiments.\nWhen purification from procyclics was attempted by immunoaffinity, clearly this did not work, but this does not mean that the SUMOylated proteins can not be purified.\nSummarizing, the Results section entitled \"Failure to purify SUMOylated proteins from T. brucei extracts\" is too long and confusing. I suggest to eliminate or substantially shorten this section, saying, as Annoura et al did, that \"failure of the detection of SUMOylated proteins after purification might be due to the rapid turnover of SUMO conjugation.\"\nValidity of the findings\nThis manuscript has a number of flaws in experimental design, and part of the results has little novelty, suggesting that its publication in its present form would be premature.\nAdditional comments\nNo comments\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"SUMOylation in Trypanosoma brucei (v0.1)\". PeerJ https://doi.org/10.7287/peerj.180v0.1/reviews/2\nReview 5: nan\nReview 6: \n",
    "LLM_extracted_review": "['Review 1: The author response to the critique is appropriate and the manuscript is acceptable for publication in PeerJ.\\n\\nReview 2: Your manuscript was reviewed by two experts and both reviewers have concerns about the presented data and request additional experiments/controls. It is critical that the major concerns identified in the critique be dealt with appropriately.\\n\\nReview 3: The paper is somewhat interesting but falls short in key originality and experimental/methodological aspects. Most of the results seem very preliminary, and additional experiments should be carried out, particularly concerning detection, identification, and validation of the potential SUMOylated proteins, and the role of SUMOylation in oxidative stress. Major issues include a lack of novelty in the data presented, failure to purify and identify any bloodstream SUMOylated protein, and unclear methodology regarding gel staining and protein detection. Minor issues include formatting errors and lack of specificity in the methods described.\\n\\nReview 4: The manuscript has a number of flaws in experimental design, and part of the results has little novelty, suggesting that its publication in its present form would be premature. Major concerns include the lack of novelty in demonstrating SUMO modification of proteins, issues with detecting the endogenous SUMOylation pattern, and the preliminary nature of RNAi experiments. The authors do not adequately demonstrate the biochemical activity of the identified proteins, and the results regarding purification of SUMOylated proteins are confusing and lack clarity. \\n\\nReview 5: No review provided.\\n\\nReview 6: No review provided.']"
}