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Systematics of the subgenus of mouse opossums Marmosa (Micoureus) (Didelphimorphia, Didelphidae) with noteworthy records from Paraguay
Mammalian Biology 77 (2012) 229–236
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Original Investigation
Systematics of the subgenus of mouse opossums Marmosa (Micoureus) (Didelphimorphia, Didelphidae) with noteworthy records from Paraguay Noé U. de la Sancha a , Guillermo D’Elía b,∗ , Pablo Teta c a
Department of Natural Resources Science, University of Rhode Island, Kingston, RI, USA Instituto de Ciencias Ambientales y Evolutivas, Universidad Austral de Chile, campus Isla Teja s/n, casilla 567, Valdivia, Chile c Unidad de Investigación Diversidad, Sistemática y Evolución, Centro Nacional Patagónico, Puerto Madryn, Chubut, Argentina b
a r t i c l e
i n f o
Article history: Received 14 July 2011 Accepted 25 October 2011 Keywords: Marmosa Marmosini Mouse-opossums South America Taxonomy
a b s t r a c t The subgenus Marmosa (Micoureus) Lesson, 1842 includes six species of long-tailed, black masked mouseopossums widely distributed in forested areas of the Neotropics from northern Argentina to Belize. Most of the nominal forms of Marmosa (Micoureus) have not been revised since 1933 and some currently accepted synonymies are in need of revision; similarly distributions of these forms remain for the most part unclear. Herein, we report Paraguayan new and noteworthy locality records for Marmosa (Micoureus), including the first records for the western Dry Chaco region. Specimens were identified to the species level on the basis of morphological and molecular data. In addition, we conducted a phylogenetic analysis that includes sequences of five of the six species currently recognized of Micoureus incorporating a total of 70 sequences of the subgenus. This constitute the most taxonomically and geographically dense phylogenetic analysis of Micoureus. Results show that the most basal dichotomy of the Micoureus clade does not delimit cis- and trans-Andean reciprocally monophyletic groups, rending the cis group paraphyletic to the single trans-species included, suggesting that the colonization of the western (trans) side of the Andes was a relatively late event in the biogeographic history of Micoureus. In addition, the phylogenetic analysis shows that additional taxonomic work is much needed to clarify the number of distinct biological units, either species or subspecies, within Micoureus. © 2011 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. All rights reserved.
Introduction The subgenus Marmosa (Micoureus) Lesson, 1842 includes six species of long-tailed, black masked mouse-opossums: M. (M.) alstoni (J.A. Allen, 1990), M. (M.) constantiae (Thomas, 1904), M. (M.) demerarae (Thomas, 1905), M. (M.) paraguayana (Tate, 1931), M. (M.) phaea (Thomas, 1899), and M. (M.) regina (Thomas, 1898). Micoureus is widely distributed in forested areas of the Neotropics from northern Argentina to Belize (Gardner and Creighton 2008; Voss and Jansa 2009). Despite recent systematic assessments (Voss et al. 2009; Rossi 2005; Rossi et al. 2010; Gutiérrez et al. 2010), most of the nominal forms of Marmosa (Micoureus) have not been revised since Tate (1933), and some currently accepted synonymies are in need of revision (Voss and Jansa 2009). As a logical consequence, distributional and ecological aspects of these species are poorly known, especially towards the southern part of the range of the genus (e.g., Flores et al. 2000). Similarly, no phylogenetic study has focused on the relationships among the species of Micoureus,
∗ Corresponding author. E-mail address: [email protected] (G. D’Elía).
hampering an understanding of the historical biogeography of the subgenus. Two species of Marmosa (Micoureus) have been recorded in Paraguay, M. (M.) constantiae Thomas, 1904 and M. (M.) paraguayana Tate, 1931. The former is only known from Parque Nacional Cerro Corá, in eastern Paraguay (Voss et al. 2009), while the second has several records throughout the eastern forested regions of this country (Brown 2004; Gardner 2005; Gardner and Creighton 2008; Krumbiegel 1941). Herein, we report new and noteworthy locality records for Marmosa (Micoureus) in Paraguay, including the first records for the western Dry Chaco region. These records are identified to the species level (cf. Tate 1931, 1933; Voss and Jansa 2009; Dias et al. 2010; Gutiérrez et al. 2010) on the basis of morphological and molecular data (including the first sequence of a Paraguayan specimen of M. (M.) paraguayana) and by comparison with the original descriptions and photographs of the holotypes. Paraguayan sequences were analyzed in the context of a phylogenetic analysis that includes sequences of five of the six species currently recognized in the subgenus Micoureus. The analyzed matrix incorporates a total of 70 sequences of Micoureus. This constitute the most taxonomically and geographically dense phylogenetic analysis of Micoureus.
1616-5047/$ – see front matter © 2011 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.mambio.2011.10.003
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Material and methods The Paraguayan records of Micoureus reported here are based on specimens housed in the Museo Nacional de Historia Natural del Paraguay (MNHNP), San Lorenzo, Paraguay; Instituto de ˜ Bioecología y Investigación Subtropical (IBIS), Pilar, Neembucú, Paraguay; The Field Museum of Natural History (FMNH), Chicago, USA; Museum of Southwestern Biology (MSB), Alburquerque, NM, USA; and Natural Science Research Laboratory (TK) and Texas Tech University, Lubbock, TX, USA. External measurements were taken from specimen labels and included, when available, total length (TL), tail (T), hind foot (HF), ear (E) and weight (W). Cranial measurements were taken with digital calipers under a dissecting microscope, following Voss et al. (2004) and included: condylobasal length (CBL), nasal breadth (NB), least interorbital breadth (LIB), zygomatic breadth (ZB), palatal length (PL), palatal breadth (PB), maxillary toothrow length (MTR), length of molars (LM), length of M1–M3 (M1–M3) and width of M4 (M4). Age classes were determined according to the criteria of Tribe (1990). Phylogenetic analyses were based on the first 801 base pairs of the mitochondrial gene that codes for cytochrome b. Sequences of 70 specimens belonging to five currently recognized species of Micoureus were analyzed (see details, including specimen and Genbank accession numbers, collection localities, and sources, in Supplementary material). Our sampling lacks sequences of M. (M.) phaea. Five sequences recovered from Paraguayan specimens were included: FMNH 211414, FMNH 211415, MSB 67000 (kindly provided by Dr. Sharon Jansa, Bell Museum of Natural History, University of Minnesota, St. Paul, USA), TK 129479, and TK 129697. In addition, we report the following five new sequences (all kindly provided by Dr. James L. Patton, Museum of Vertebrate Zoology, University of California, Berkeley, USA) recovered from specimens of M. (M.) alstoni (FMG2281, FMG2305, USNM449565), M. (M.) paraguayana (MZUSP 29195), and M. (M.) regina (MVZ 190324). All new sequences were deposited in Genbank (accession numbers: JN887133–42). DNA sequences gathered by us were acquired ˜ (2004). Hapfollowing the protocol outlined in D’Elía and Pardinas lotypes recovered from three species of Marmosa (Marmosa) were used to conform the outgroup: M. (Ma.) lepida (HM106377), M. (Ma.) mexicana (HM106357) and M. (Ma.) murina (HM106369). Sequences were aligned using Clustal X (Thompson et al. 1997) with default values for all alignment parameters; no adjustment by eye was needed. Observed percentage of sequence divergence was calculated with MEGA 5 (Tamura et al., 2011) ignoring those sites with missing data. Sequence alignment was subjected to maximum parsimony (MP; Farris 1982) and Bayesian (Huelsenbeck et al. 2001) analyses. Characters used in MP analysis were treated as unordered and equally weighted. MP was conducted in PAUP* (Swofford 2000) with 500 replicates of heuristic search with random addition of sequences and TBR branch swapping. Nodal support was assessed with 1000 pseudoreplicates of Bootstrap (BS; Felsenstein 1985), each with five replicates of random addition of sequences and TBR branch swapping. Bayesian analysis was conducted with MrBayes 3 (Ronquist and Huelsenbeck 2003) by means of two independent runs with three heated and one cold Markov chains each. A model with six categories of base substitution, a gamma-distributed rate parameter, and a proportion of invariant sites was specified; all model parameters were estimated in MrBayes. Uniform interval priors were assumed for all parameters except base composition and GTR parameters, which assumed a Dirichlet process prior. Chains were run for 10 million generations and trees were sampled every 1000 generations for each chain. Log-likelihood values were plotted against generation time for each run to check that each converged on a stable log-likelihood value. The first 25% of the trees were discarded as burn-in; remaining trees were used to compute a 50% majority rule
Table 1 Observed genetic distance of the cytochrome b gene within and among five species of Marmosa (Micoureus). Numbers between parentheses refer to the number of sequences studied for each species. The sample of M. (M.) demerarae does not include haplotype U34673; see text for details.
alstoni (3) constantiae (3) demerarae (35) paraguayanus (23) regina (5)
Intraspecific
Interspecific
0.000 0.010 0.037 0.020 0.017
0.132 0.091 0.097 0.126
0.140 0.113 0.083
0.091 0.131
0.119
consensus tree and obtain posterior probability (PP) estimates for each clade. Results Levels of observed intraspecific variation range from 0 to 3.7% (alstoni and demerarae respectively (Table 1). Meanwhile, interspecific comparisons reach large values in all cases (Table 1); values range from 8.3 (constantiae vs. regina) to 13.2% (alstoni vs. constantiae). Maximum parsimony analysis recovered 640 shortest trees (789 steps; consistency index = 0.530; retention index = 0.874). The topology of the strict consensus of these trees is fully congruent with that found in the Bayesian analysis (Fig. 1). All species, but demerarae, are recovered monophyletic. One haplotype (Genbank accession number U34673) assigned to demerarae appears as sister to alstoni. Haplotype U34673 is highly divergent from all others studied (9.5% respect the alstoni clade; 10.3% respect to the demerarae clade). The Micoureus clade is well supported (BS = 99; PP = 1) and relationships among species are fully resolved. The basal dichotomy of the clade of Micoureus leaves to a clade (BS = 100; PP = 1) formed by regina and constantiae in one hand and to another clade (BS = 90; PP = 0.96) formed by paraguayana sister to the clade (demerarae (haplotype U34673, alstoni)) in the other hand. Placement of haplotypes recovered from Paraguayan specimens (Fig. 2) is unambiguous and congruent with morphology (see below): MSB 67000 falls in the constantiae clade; while FMNH 211414, FMNH 211415, TK 129479, and TK 129697 fall in the paraguayana clade. Specimens of M. (M.) constantiae and M. (M.) paraguayana can be easily discriminated by the color of the ventral fur, which is buffy to orangish, mostly gray-based in paraguayana and yellowish to creamy white, not gray based, in constantiae (Gardner, 2008). In addition, the dorsal coloration of constantiae is gray with a slight cast of brownish or yellowish, while paraguayana is house mouse-gray with no admixture of brown (Tate, 1933). The skull of constantiae is large, with broad zygomatic arches, well-developed and triangular postorbital process, a well-marked postorbital constriction, and pronounced lambdoidal crests and temporal ridges. Meanwhile, the skull of paraguayana lack sharp or well developed postorbital processes and a defined postorbital constriction; instead there are well developed supraorbital beads, in adults, that continue posteriorly as parallel ridges across the parietal bones (Fig. 3). New locality records and some taxonomic issues of Paraguayan examples of Marmosa (Micoureus) are presented (Table 2). Marmosa (Micoureus) constantiae New records (codes between brackets correspond to those of Fig. 2): Alto Paraguay: Cerro León, Parque Nacional Defensores del Chaco [PY1], -20.360 -60.450 (MNHNP 0481, 1659, 1660); Puerto Casado [PY2], -22.333 -57.917 (FMNH 54404); Boquerón: Km 155 of Trans Chaco (road) [PY3], -23.500 -59.700 (MNHNP 121795); Amambay: 33 km SE Pedro Juan Caballero [PY4], -22.741 -55.680 (MSB 67000).
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Fig. 1. Genealogical relationship of 70 haplotypes of the subgenus Marmosa (Micoureus). Bayesian posterior probabilities (>0.50) indicating nodal support are shown next to nodes. Numbers within parentheses indicate localities from where haplotypes were recovered (Fig. 4 and Supplementary material).
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smaller than those from the Cerrado (Fig. 3), but our low sample size prevents any statistical evaluation of this point. This is not a minor issue, because the main differences between M. constantatiae constantiae (with type locality in “Chapada”, Matto Grosso, Brazil; Thomas, 1904: 243) and M. constantiae budini (“Altura de Yuto, Río San Francisco”, Jujuy, Argentina; Thomas, 1920: 195), as described by Tate (1933; see also Anderson 1997), is just size, with the latter being smaller. However, as mentioned by Tate (1933) both nominal forms may ultimately be merged as one entity. The analysis of a larger sample including a phylogeographic analysis would help in resolving this issue. Contreras (1984) reported a specimen identified as Marmosa cinerea paraguayana (C-001182; housed at the Instituto de Bioecología y Investigación Subtropical, Pilar, Paraguay) from Puerto Bouvier [AR11] (-25.450, -57.583, Departamento Pilcomayo, Formosa Province, Argentina), although the specimen tag reads “Clorinda, Formosa”. Examination of this specimen (by NDLS) revealed that it is a misidentified individual assignable to M. (M.) constantiae. This finding is the easternmost record of this species in Argentina and the only known Argentinean collection locality outside the Yungas, enlarging the known distributional range of this mouse-opossum in the country more than 770 km to the E. This record is about 280 km ESE from the closest Paraguayan Dry Chaco record. Our report bridges a large gap in the previously known distribution of M. (M.) constantiae, a species whose distribution appears to mirror that of the didelphids Monodelphis kunsi (see de la Sancha et al., 2007) and M. domestica (see Pine and Handley, 2008).
Marmosa (Micoureus) paraguayana Fig. 2. Recording localities for the specimens of Marmosa (Micoureus) constantiae (triangles) and M. (M.) paraguayana in Paraguay and one Argentine locality. The star corresponds to the type locality of Marmosa cinerea paraguayana Tate, 1931. For locality information see the text.
Comments: This species has been previously reported from lower elevations in southeastern Bolivia, southwestern Brazil, and the northwestern Yungas of Argentina (Gardner and Creighton 2008). The new locality records fill in the major gaps between the western Brazilian and the northeastern Argentinean records and thus expand its known distribution by more than 530 km to the SW (Fig. 2). Specimens from the Dry Chaco appear to be slightly
New records (codes between brackets correspond to those of Fig. 2): Canindeyú: Colonia Britez Cue [PY5], -24.239 -55.281 (MNHNP 141262), 3.3 km N Curuguaty [PY6], -24.517 -55.700 (UMMZ 134551); Caaguazú: Reserva Morombi [PY7], -24.715 55.433 (FMNH 211416), -24.713, -55.438, (TK129697); Alto Paraná: Reserva Limoy [PY8], -24.730 -54.400 (211414); Hernandarias [PY9], -25.370 -54.750 (MIB 32); Itapúa: Reserva de Recursos y Manejo San Rafael [PY10], -26.574 -55.680 (FMNH 211415). Comments: All specimens herein assigned by us to paraguayana fit the original description by Tate (1931) of Marmosa cinerea paraguayana, both in coloration as well in external and cranial
Fig. 3. Cranial comparisons (dorsal, ventral, and lateral views of skull and labial view of the mandible), from left to right, of Marmosa (Micoureus) constantiae [MNHNP 0481 (Dry Chaco), MSB 67000 (Cerrado)], and M. (M.) paraguayana [FMNH 129479, (Atlantic Forest)] from Paraguay. Scale = 20 mm.
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Table 2 External and cranial measurements of studied specimens of Marmosa (Micoureus) from Paraguay (see the text for the acronyms explanation). All measurements are in millimeters and were taken following Voss et al. (2004); weight is reported in grams. Age classes are based on Tribe (1990). Specimen
Age
MNHNP 1659 MNHNP 1660 MNHNP 0481 MNHNP 121795 MNHNP 121753 FMNH 54404 MSB 67000 UMMZ 134550 UMMZ 134551 FMNH 211414 FMNH 211415 FMNH 211416 MNHNP 141262 MNHNP 121753 TK 129479
– – 6 7 5 – 6 4 6 3 5 6 – – 5
W 50 30 57 73 – – 90.5 – – 26 58 140 663 46 83
TL
T
HF
E
220 305 352 412 – 247 385 – – 275 338 400 335 356 396
135 145 213 213 – 150 235 – – 169 202 221 192 206 230
25 21 26 28 – 20 29 – – 21 24 26 27 25 26
23 20 23 27 – 12 17 – – 21 24 29 24 26 27
ZB
LIB
NB
CBL
PL
PB
MTL
LM
M1-3
M4
– 20.7 – 21.15 – 23.54 20.58 24.24 17.24 23.03 23.38 – – 21.29
– – 7 – 7.61 – 6.16 6.16 7.43 6.21 7.16 8.35 – – 6.87
– – – – 5.38 – 4.78 4.08 5.46 2.85 3.76 5.67 – – 4.09
– – 37 – 37.42 – 40.01 34.51 41.63 31.02 40.7 40.24 – – 36.73
– – 20.5 – 20.96 – 21.02 18.91 22.84 17.25 22.85 22.44 – – 20.59
– – 12.2 – 13.94 – 13.57 13.13 14.12 11.47 14.14 14.21 – – 13.6
– – 15.5 – 16.86 – 16.49 15.27 17.69 12.47 17.6 17.36 – – 16.68
– – 7.7 – 8.85 – 8.39 8.54 9.05 – 9.24 9.21 – – 9.12
– – 6.6 – 7.66 – 7.13 7.29 7.74 7.23 7.91 7.85 – – 7.54
– – – – 2.83 – 2.84 2.8 2.85 – 3.07 3.13 – – 2.79
measurements and characteristics (Tate 1931; Fig. 2; see above). New locality records fill the gap between previously known localities, showing that this species is widely distributed throughout the eastern forested portion of Paraguay (Fig. 2). In addition, our molecular data, including the first haplotypes from near the type locality (“Villa Rica, Paraguay”; Tate 1931: 1), suggest that only one form of Marmosa (Micoureus) is found and widely dispersed in eastern Paraguay, northeastern Argentina, and southeastern Brazil.
Discussion The phylogenetic analysis shows that the two main lineages of Micoureus are present in Paraguay. One of this is represented by M. (M.) paraguayana that inhabits the forested areas of eastern Paraguay. Meanwhile, the other main lineage is represented by M. (M.) constantiae, which is distributed in open areas of both Chaco and the Cerrado in eastern Paraguay. This finding highlights the diversity of Paraguay not just at the species level but also phylogenetically. This is important given that the mammalian fauna of Paraguay is one of the least known in South America (Pine 1982; Myers et al. 2002) even the long history of reports about these vertebrates (de Azara 1801, 1802; Rengger 1830; Bertoni 1914). This scenario is reflected in several recent additions to the known mammal fauna of Paraguay, which include a marsupial (de la Sancha et al. 2007), a bat (Stevens et al. 2010), several rodents including a new family (D‘Elía et al. 2008; Percequillo et al. 2008; de la Sancha et al. 2009a, 2011), an armadillo (Smith et al., in press), and even an exotic lagomorph (de la Sancha et al. 2009b). Additional field work will aid in improving sample sizes as well as providing much needed taxonomic, ecological, and distributional data about these groups. This is especially urgent in bioregions like the Interior Atlantic Forest which are quickly disappearing (Huang et al. 2007, 2009). Relationships among species of Micoureus are fully resolved (Fig. 1). Interestingly, the most basal dichotomy of the Micoureus clade does not delimit cis- and trans-Andean (sensu Haffer 1967) reciprocally monophyletic groups. M. (M.) alstoni the single transAndean species included in the analysis is well nested in the Micoureus clade; i.e., cis-Andean species form a paraphyletic group with respect to the trans-Andean species (Fig. 4). This result suggests that the colonization of the western (trans) side of the Andes would be a relatively late event in the biogeographic history of Micoureus. In this respect, is of much interest to include in the phylogenetic analysis representatives of M. (M.) phaea, the other trans-Andean species of the subgenus (Fig. 4), to clarify if it crossed the Andes on a single event or if the western side of the Andes was colonized more than once by Micoureus.
Our sampling of paraguayana includes 23 haplotypes, which allows for a preliminary picture of the geographic structure of the genetic variation of this species. The species has genealogical structure but it is not geographically structured (Fig. 1). These results support Gardner and Creighton (2008) whom do not recognize subspecies within paraguayana. Paraguayan haplotypes are well nested in the paraguayana clade rendering the mostly coastal Brazilian sample paraphyletic to Paraguayan variants. A similar pattern was found in the analysis of a small sample of Akodon paranaensis (D‘Elía et al., 2008). In light of this result we suggest that M. (M.) paraguayana would have colonized eastern Paraguay from a source in the Atlantic coastal region. A similar demographic scenario is being suggested for the sigmodontine mice Akodon montensis in association with Atlantic Forest dynamics during the Pleistocene (Valdez and D‘Elía, in prep.; see also Carnaval and Moritz, 2008). Finally, the genealogical analysis shows that additional taxonomic work is needed to clarify the number of distinct biological units, either species or subspecies, within Micoureus. For example, M. (M.) demerarae was not recovered as monophyletic, given that one haplotype (GenBank accession number U34673) assigned to demerarae appears as sister to alstoni. A similar result was already obtained by Dias et al. (2010) with a reduced taxonomic sampling. Haplotype U34673 is highly divergent from all others studied (9.5% respect the alstoni clade; 10.3% respect to the demerarae clade), raising the possibility that the specimen from which it was recovered represents a distinct species of those studied here. Since we have not had the opportunity to study the voucher, we will not proceed further on this issue. In addition to the issue raised by the position of haplotype U34673, results show that M. (M.) regina and M. (M.) demerarae present deep phylogeographic structure. M. (M.) regina shows two strongly supported allopatric clades (Fig. 1) that differ on average by 2.7%. However, sampling is scarce precluding the evaluation of the congruence between this phylogeographic break and the subpecies recognized by Gardner and Creighton (2008). Three main allopatric clades were found for M. (M.) demerarae that, on average, differ among them by 5.0% (Fig. 4b). One clade (1; intraclade variation: 1.9%) distributes in northern Brazil (Amazonas), southern Venezuela, Guyana and French Guiana; clade 1 in turns divides into two subclades: 1a in northern Brazil (Amazonas), and southern Venezuela and 1b in Guyana and French Guiana. The second main clade (2; intraclade variation: 2.5%), sister to clade 1, distributes in central and eastern Brazil (Bahia, Mato Groso, and Tocantins); similarly clade 2 is composed of three subclades that are geographically segregated in the longitudinal dimension. The third main clade (3; intraclade variation: 1.7%) of M. (M.) demerarae, sister to the clade formed by clades 1 and 2, distributes in
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Fig. 4. Distribution of the currently recognized species of Micoureus taken from Gardner and Creighton (2008) and modified according the results of the present study. Localities from were specimens were taken for the phylogenetic analysis are shown; locality numbers are those used in Fig. 1 (see also main text and Supplementary material). (A) Phylogenetic relationships among species of Micoureus as recovered in the cytochrome b gene sequence analysis (see text and Fig. 1 for reference numbers). (B) Phylogenetic relationships among the phylogeographic units found within M. (M.) demerarae. Type localities of the five currently recognized subspecies of M. (M.) demerarae are indicated with gray circles, while those of the two taxa currently considered synonyms of recognized subspecies are indicated with gray circles with a black cross. Taxonomy follows Gardner and Creighton (2008).
eastern Peru and central Brazil (Mato Groso). Analyzing other data sets Costa (2003) and Patton and Costa (2003) found similar results. Even when, as those of Costa (2003) and Patton and Costa (2003), our sampling is geographically partial and does not include any topogenetype (sensu Chakrabarty, 2010), it is clear that the phylogeographic pattern of M. (M.) demerarae is not fully concordant with the subspecific arrangement recognized by Gardner and Creighton (2008) in which five subspecies are considered. For example, as already mentioned by Gardner and Creighton (2008) the phylogeographic unit of Peru and southwestern Brazil (clade 3) apparently lacks a name. However, we note that the type locality of dominus Thomas, 1920, a name currently assigned to populations in eastern
Brazil (i.e., clade 2), is geographically closer to sampled populations of the northern (1) and western (3) clades than to those of clade 2. Therefore, further geographic sampling may show that dominus may in fact apply to the western phylogeographic unit or the northern clade (in which case would be a synomyn of nominotypical subspecies) and not to the eastern one. Then, if proved that dominus applies to either clade 1 or 3, limae Thomas, 1920, a form currently considered a synomyn of dominus and with type locality laying close to the geographic distributional area of the eastern clade (2), may be the correct name to apply to it. The important issue going beyond these taxonomic scenarios and the one that we want to highlight here is that these results, once again, reinforce
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the long standing claim that further basic research involving field collection of additional specimen series and museum based work is still much needed. Acknowledgements We thank Sharon Jansa and Jim Patton whom generously provided unpublished data and Ronald Pine, Alfred Gardner, Shayna Harris, and an anonymous reviewer for comments and revisions of this manuscript. The assistance of Robert Baker and Heath Garder (TTU), Bruce Patterson (FMNH), Phil Myers (UMMZ), Joe Cook and Jon Dunnum (MSB) and the staff at the Museo Nacional de Historia Natural del Paraguay is greatly appreciated. We thank Tom Husband and the Conservation Genetics Laboratoy at University of Rhode Island (URI) where several sequences were generated. This research is based in part upon work conducted using the Rhode Island Genomics and Sequencing Center which is supported in part by the National Science Foundation (MRI Grant DBI-0215393 and EPSCoR Awards 0554548 & 1004057), the US Department of Agriculture (Grants 2002-34438-12688, 2003-34438-13111 and 2008-34438-19246), and the URI. Financial support to NDLS was provided by American Philosophical Society (through the Lewis and Clark Exploration Fund), the Marshall Field Collection Fund of the Field Museum of Natural History in Chicago, the TTU Association of Biologists (TTUAB) mini-grant, the TTU Graduate School, for a AT&T/McNair Fellowship, assistantships from the Department of Biological Sciences (TTU), the Mary Rice Foundation (special thanks to Mrs. Mary Rice), the International Institute for Education (via a Fulbright Scholarship), a Michelle Knapp Memorial Scholarship (TTU), and a Knox Jones Award (TTU). FONDECYT 1110737 and MECESUP AUS0805 provided support to GD. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mambio.2011.10.003. References Anderson, S., 1997. Mammals of Bolivia: taxonomy and distribution. Bull. Am. Mus. Nat. Hist. 231, 1–652. de Azara, F., 1801. Essais sur lˇıhistoire naturelle des quadrupèdes de la Province du Paraguay. Tome Second. Imprière C. Pougens, Paris. de Azara, F., 1802. Apuntamientos para la historia natural de los quadrúpedos del Paraguay y Río de La Plata. Tomo segundo. Imprenta de la Viuda de Ibarra, Madrid. Bertoni, A.de W., 1914. Fauna paraguaya: catálogos sistemáticos de los vertebrados del Paraguay: peces, batracios, reptiles, aves, y mamíferos conocidos hasta 1913 Asunción: Establecimiento Gráfico M. Brossa. Descripción física y económica del Paraguay. Numeración Novenal 59, 1–86. Brown, E.B., 2004. Atlas of New World marsupials. Fieldiana Zool. 102, 1–308. Carnaval, A.C., Moritz, C., 2008. Historical climate modelling predicts patterns of current biodiversity in the Brazilian Atlantic forest. J. Biogeogr. 35, 1187–1201. Chakrabarty, P., 2010. Genetypes: a concept to help integrate molecular phylogenetics and taxonomy. Zootaxa 2632, 67–68. Contreras, J.P., 1984. Notas sobre el género Marmosa en el noreste Argentino (Marsupialia: Didelphidae). Hist. Nat. 4, 11–12. Costa, L.P., 2003. The historical bridge between the Amazon and Atlantic Forest of Brazil: a study of molecular phylogeography with small mammals. J. Biogeogr. 30, 71–86. de la Sancha, N.U., D’Elía, G., Tribe, C.J., Perez, P.E., Valdez, L., Pine, R.H., 2011. Rhipidomys (Rodentia, Cricetidae) from Paraguay: noteworthy new records and identity of the Paraguayan species. Mammalia 75, 269–276. de la Sancha, N.U., Mantilla-Meluk, H., Ramirez, F., Perez, P., Mujica, N., Troche, A., Giménez, M., 2009b. Notes on geographic distribution: Mammalia, Lagomorpha, Leporidae, Lepus europaeus, Pallas, 1778: distribution extension, first confirmed record for Paraguay. Check List 5, 428–432. de la Sancha, N., D‘Elía, G., Netto, F., Perez, P., Salazar-Bravo, J., 2009a. Discovery of Juliomys (Rodentia, Sigmodontinae) in Paraguay, a new genus of Sigmodontinae for the country’s Atlantic Forest. Mammalia 73, 162–167. de la Sancha, N., Solari, S., Owen, R.D., 2007. First records of Monodelphis kunsi Pine (Didelphimorphia, Didelphidae) from Paraguay, with an evaluation of its distribution. Mastozool. Neotrop. 14, 241–247.
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Original Investigation
Systematics of the subgenus of mouse opossums Marmosa (Micoureus) (Didelphimorphia, Didelphidae) with noteworthy records from Paraguay Noé U. de la Sancha a , Guillermo D’Elía b,∗ , Pablo Teta c a
Department of Natural Resources Science, University of Rhode Island, Kingston, RI, USA Instituto de Ciencias Ambientales y Evolutivas, Universidad Austral de Chile, campus Isla Teja s/n, casilla 567, Valdivia, Chile c Unidad de Investigación Diversidad, Sistemática y Evolución, Centro Nacional Patagónico, Puerto Madryn, Chubut, Argentina b
a r t i c l e
i n f o
Article history: Received 14 July 2011 Accepted 25 October 2011 Keywords: Marmosa Marmosini Mouse-opossums South America Taxonomy
a b s t r a c t The subgenus Marmosa (Micoureus) Lesson, 1842 includes six species of long-tailed, black masked mouseopossums widely distributed in forested areas of the Neotropics from northern Argentina to Belize. Most of the nominal forms of Marmosa (Micoureus) have not been revised since 1933 and some currently accepted synonymies are in need of revision; similarly distributions of these forms remain for the most part unclear. Herein, we report Paraguayan new and noteworthy locality records for Marmosa (Micoureus), including the first records for the western Dry Chaco region. Specimens were identified to the species level on the basis of morphological and molecular data. In addition, we conducted a phylogenetic analysis that includes sequences of five of the six species currently recognized of Micoureus incorporating a total of 70 sequences of the subgenus. This constitute the most taxonomically and geographically dense phylogenetic analysis of Micoureus. Results show that the most basal dichotomy of the Micoureus clade does not delimit cis- and trans-Andean reciprocally monophyletic groups, rending the cis group paraphyletic to the single trans-species included, suggesting that the colonization of the western (trans) side of the Andes was a relatively late event in the biogeographic history of Micoureus. In addition, the phylogenetic analysis shows that additional taxonomic work is much needed to clarify the number of distinct biological units, either species or subspecies, within Micoureus. © 2011 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. All rights reserved.
Introduction The subgenus Marmosa (Micoureus) Lesson, 1842 includes six species of long-tailed, black masked mouse-opossums: M. (M.) alstoni (J.A. Allen, 1990), M. (M.) constantiae (Thomas, 1904), M. (M.) demerarae (Thomas, 1905), M. (M.) paraguayana (Tate, 1931), M. (M.) phaea (Thomas, 1899), and M. (M.) regina (Thomas, 1898). Micoureus is widely distributed in forested areas of the Neotropics from northern Argentina to Belize (Gardner and Creighton 2008; Voss and Jansa 2009). Despite recent systematic assessments (Voss et al. 2009; Rossi 2005; Rossi et al. 2010; Gutiérrez et al. 2010), most of the nominal forms of Marmosa (Micoureus) have not been revised since Tate (1933), and some currently accepted synonymies are in need of revision (Voss and Jansa 2009). As a logical consequence, distributional and ecological aspects of these species are poorly known, especially towards the southern part of the range of the genus (e.g., Flores et al. 2000). Similarly, no phylogenetic study has focused on the relationships among the species of Micoureus,
∗ Corresponding author. E-mail address: [email protected] (G. D’Elía).
hampering an understanding of the historical biogeography of the subgenus. Two species of Marmosa (Micoureus) have been recorded in Paraguay, M. (M.) constantiae Thomas, 1904 and M. (M.) paraguayana Tate, 1931. The former is only known from Parque Nacional Cerro Corá, in eastern Paraguay (Voss et al. 2009), while the second has several records throughout the eastern forested regions of this country (Brown 2004; Gardner 2005; Gardner and Creighton 2008; Krumbiegel 1941). Herein, we report new and noteworthy locality records for Marmosa (Micoureus) in Paraguay, including the first records for the western Dry Chaco region. These records are identified to the species level (cf. Tate 1931, 1933; Voss and Jansa 2009; Dias et al. 2010; Gutiérrez et al. 2010) on the basis of morphological and molecular data (including the first sequence of a Paraguayan specimen of M. (M.) paraguayana) and by comparison with the original descriptions and photographs of the holotypes. Paraguayan sequences were analyzed in the context of a phylogenetic analysis that includes sequences of five of the six species currently recognized in the subgenus Micoureus. The analyzed matrix incorporates a total of 70 sequences of Micoureus. This constitute the most taxonomically and geographically dense phylogenetic analysis of Micoureus.
1616-5047/$ – see front matter © 2011 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.mambio.2011.10.003
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Material and methods The Paraguayan records of Micoureus reported here are based on specimens housed in the Museo Nacional de Historia Natural del Paraguay (MNHNP), San Lorenzo, Paraguay; Instituto de ˜ Bioecología y Investigación Subtropical (IBIS), Pilar, Neembucú, Paraguay; The Field Museum of Natural History (FMNH), Chicago, USA; Museum of Southwestern Biology (MSB), Alburquerque, NM, USA; and Natural Science Research Laboratory (TK) and Texas Tech University, Lubbock, TX, USA. External measurements were taken from specimen labels and included, when available, total length (TL), tail (T), hind foot (HF), ear (E) and weight (W). Cranial measurements were taken with digital calipers under a dissecting microscope, following Voss et al. (2004) and included: condylobasal length (CBL), nasal breadth (NB), least interorbital breadth (LIB), zygomatic breadth (ZB), palatal length (PL), palatal breadth (PB), maxillary toothrow length (MTR), length of molars (LM), length of M1–M3 (M1–M3) and width of M4 (M4). Age classes were determined according to the criteria of Tribe (1990). Phylogenetic analyses were based on the first 801 base pairs of the mitochondrial gene that codes for cytochrome b. Sequences of 70 specimens belonging to five currently recognized species of Micoureus were analyzed (see details, including specimen and Genbank accession numbers, collection localities, and sources, in Supplementary material). Our sampling lacks sequences of M. (M.) phaea. Five sequences recovered from Paraguayan specimens were included: FMNH 211414, FMNH 211415, MSB 67000 (kindly provided by Dr. Sharon Jansa, Bell Museum of Natural History, University of Minnesota, St. Paul, USA), TK 129479, and TK 129697. In addition, we report the following five new sequences (all kindly provided by Dr. James L. Patton, Museum of Vertebrate Zoology, University of California, Berkeley, USA) recovered from specimens of M. (M.) alstoni (FMG2281, FMG2305, USNM449565), M. (M.) paraguayana (MZUSP 29195), and M. (M.) regina (MVZ 190324). All new sequences were deposited in Genbank (accession numbers: JN887133–42). DNA sequences gathered by us were acquired ˜ (2004). Hapfollowing the protocol outlined in D’Elía and Pardinas lotypes recovered from three species of Marmosa (Marmosa) were used to conform the outgroup: M. (Ma.) lepida (HM106377), M. (Ma.) mexicana (HM106357) and M. (Ma.) murina (HM106369). Sequences were aligned using Clustal X (Thompson et al. 1997) with default values for all alignment parameters; no adjustment by eye was needed. Observed percentage of sequence divergence was calculated with MEGA 5 (Tamura et al., 2011) ignoring those sites with missing data. Sequence alignment was subjected to maximum parsimony (MP; Farris 1982) and Bayesian (Huelsenbeck et al. 2001) analyses. Characters used in MP analysis were treated as unordered and equally weighted. MP was conducted in PAUP* (Swofford 2000) with 500 replicates of heuristic search with random addition of sequences and TBR branch swapping. Nodal support was assessed with 1000 pseudoreplicates of Bootstrap (BS; Felsenstein 1985), each with five replicates of random addition of sequences and TBR branch swapping. Bayesian analysis was conducted with MrBayes 3 (Ronquist and Huelsenbeck 2003) by means of two independent runs with three heated and one cold Markov chains each. A model with six categories of base substitution, a gamma-distributed rate parameter, and a proportion of invariant sites was specified; all model parameters were estimated in MrBayes. Uniform interval priors were assumed for all parameters except base composition and GTR parameters, which assumed a Dirichlet process prior. Chains were run for 10 million generations and trees were sampled every 1000 generations for each chain. Log-likelihood values were plotted against generation time for each run to check that each converged on a stable log-likelihood value. The first 25% of the trees were discarded as burn-in; remaining trees were used to compute a 50% majority rule
Table 1 Observed genetic distance of the cytochrome b gene within and among five species of Marmosa (Micoureus). Numbers between parentheses refer to the number of sequences studied for each species. The sample of M. (M.) demerarae does not include haplotype U34673; see text for details.
alstoni (3) constantiae (3) demerarae (35) paraguayanus (23) regina (5)
Intraspecific
Interspecific
0.000 0.010 0.037 0.020 0.017
0.132 0.091 0.097 0.126
0.140 0.113 0.083
0.091 0.131
0.119
consensus tree and obtain posterior probability (PP) estimates for each clade. Results Levels of observed intraspecific variation range from 0 to 3.7% (alstoni and demerarae respectively (Table 1). Meanwhile, interspecific comparisons reach large values in all cases (Table 1); values range from 8.3 (constantiae vs. regina) to 13.2% (alstoni vs. constantiae). Maximum parsimony analysis recovered 640 shortest trees (789 steps; consistency index = 0.530; retention index = 0.874). The topology of the strict consensus of these trees is fully congruent with that found in the Bayesian analysis (Fig. 1). All species, but demerarae, are recovered monophyletic. One haplotype (Genbank accession number U34673) assigned to demerarae appears as sister to alstoni. Haplotype U34673 is highly divergent from all others studied (9.5% respect the alstoni clade; 10.3% respect to the demerarae clade). The Micoureus clade is well supported (BS = 99; PP = 1) and relationships among species are fully resolved. The basal dichotomy of the clade of Micoureus leaves to a clade (BS = 100; PP = 1) formed by regina and constantiae in one hand and to another clade (BS = 90; PP = 0.96) formed by paraguayana sister to the clade (demerarae (haplotype U34673, alstoni)) in the other hand. Placement of haplotypes recovered from Paraguayan specimens (Fig. 2) is unambiguous and congruent with morphology (see below): MSB 67000 falls in the constantiae clade; while FMNH 211414, FMNH 211415, TK 129479, and TK 129697 fall in the paraguayana clade. Specimens of M. (M.) constantiae and M. (M.) paraguayana can be easily discriminated by the color of the ventral fur, which is buffy to orangish, mostly gray-based in paraguayana and yellowish to creamy white, not gray based, in constantiae (Gardner, 2008). In addition, the dorsal coloration of constantiae is gray with a slight cast of brownish or yellowish, while paraguayana is house mouse-gray with no admixture of brown (Tate, 1933). The skull of constantiae is large, with broad zygomatic arches, well-developed and triangular postorbital process, a well-marked postorbital constriction, and pronounced lambdoidal crests and temporal ridges. Meanwhile, the skull of paraguayana lack sharp or well developed postorbital processes and a defined postorbital constriction; instead there are well developed supraorbital beads, in adults, that continue posteriorly as parallel ridges across the parietal bones (Fig. 3). New locality records and some taxonomic issues of Paraguayan examples of Marmosa (Micoureus) are presented (Table 2). Marmosa (Micoureus) constantiae New records (codes between brackets correspond to those of Fig. 2): Alto Paraguay: Cerro León, Parque Nacional Defensores del Chaco [PY1], -20.360 -60.450 (MNHNP 0481, 1659, 1660); Puerto Casado [PY2], -22.333 -57.917 (FMNH 54404); Boquerón: Km 155 of Trans Chaco (road) [PY3], -23.500 -59.700 (MNHNP 121795); Amambay: 33 km SE Pedro Juan Caballero [PY4], -22.741 -55.680 (MSB 67000).
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Fig. 1. Genealogical relationship of 70 haplotypes of the subgenus Marmosa (Micoureus). Bayesian posterior probabilities (>0.50) indicating nodal support are shown next to nodes. Numbers within parentheses indicate localities from where haplotypes were recovered (Fig. 4 and Supplementary material).
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smaller than those from the Cerrado (Fig. 3), but our low sample size prevents any statistical evaluation of this point. This is not a minor issue, because the main differences between M. constantatiae constantiae (with type locality in “Chapada”, Matto Grosso, Brazil; Thomas, 1904: 243) and M. constantiae budini (“Altura de Yuto, Río San Francisco”, Jujuy, Argentina; Thomas, 1920: 195), as described by Tate (1933; see also Anderson 1997), is just size, with the latter being smaller. However, as mentioned by Tate (1933) both nominal forms may ultimately be merged as one entity. The analysis of a larger sample including a phylogeographic analysis would help in resolving this issue. Contreras (1984) reported a specimen identified as Marmosa cinerea paraguayana (C-001182; housed at the Instituto de Bioecología y Investigación Subtropical, Pilar, Paraguay) from Puerto Bouvier [AR11] (-25.450, -57.583, Departamento Pilcomayo, Formosa Province, Argentina), although the specimen tag reads “Clorinda, Formosa”. Examination of this specimen (by NDLS) revealed that it is a misidentified individual assignable to M. (M.) constantiae. This finding is the easternmost record of this species in Argentina and the only known Argentinean collection locality outside the Yungas, enlarging the known distributional range of this mouse-opossum in the country more than 770 km to the E. This record is about 280 km ESE from the closest Paraguayan Dry Chaco record. Our report bridges a large gap in the previously known distribution of M. (M.) constantiae, a species whose distribution appears to mirror that of the didelphids Monodelphis kunsi (see de la Sancha et al., 2007) and M. domestica (see Pine and Handley, 2008).
Marmosa (Micoureus) paraguayana Fig. 2. Recording localities for the specimens of Marmosa (Micoureus) constantiae (triangles) and M. (M.) paraguayana in Paraguay and one Argentine locality. The star corresponds to the type locality of Marmosa cinerea paraguayana Tate, 1931. For locality information see the text.
Comments: This species has been previously reported from lower elevations in southeastern Bolivia, southwestern Brazil, and the northwestern Yungas of Argentina (Gardner and Creighton 2008). The new locality records fill in the major gaps between the western Brazilian and the northeastern Argentinean records and thus expand its known distribution by more than 530 km to the SW (Fig. 2). Specimens from the Dry Chaco appear to be slightly
New records (codes between brackets correspond to those of Fig. 2): Canindeyú: Colonia Britez Cue [PY5], -24.239 -55.281 (MNHNP 141262), 3.3 km N Curuguaty [PY6], -24.517 -55.700 (UMMZ 134551); Caaguazú: Reserva Morombi [PY7], -24.715 55.433 (FMNH 211416), -24.713, -55.438, (TK129697); Alto Paraná: Reserva Limoy [PY8], -24.730 -54.400 (211414); Hernandarias [PY9], -25.370 -54.750 (MIB 32); Itapúa: Reserva de Recursos y Manejo San Rafael [PY10], -26.574 -55.680 (FMNH 211415). Comments: All specimens herein assigned by us to paraguayana fit the original description by Tate (1931) of Marmosa cinerea paraguayana, both in coloration as well in external and cranial
Fig. 3. Cranial comparisons (dorsal, ventral, and lateral views of skull and labial view of the mandible), from left to right, of Marmosa (Micoureus) constantiae [MNHNP 0481 (Dry Chaco), MSB 67000 (Cerrado)], and M. (M.) paraguayana [FMNH 129479, (Atlantic Forest)] from Paraguay. Scale = 20 mm.
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Table 2 External and cranial measurements of studied specimens of Marmosa (Micoureus) from Paraguay (see the text for the acronyms explanation). All measurements are in millimeters and were taken following Voss et al. (2004); weight is reported in grams. Age classes are based on Tribe (1990). Specimen
Age
MNHNP 1659 MNHNP 1660 MNHNP 0481 MNHNP 121795 MNHNP 121753 FMNH 54404 MSB 67000 UMMZ 134550 UMMZ 134551 FMNH 211414 FMNH 211415 FMNH 211416 MNHNP 141262 MNHNP 121753 TK 129479
– – 6 7 5 – 6 4 6 3 5 6 – – 5
W 50 30 57 73 – – 90.5 – – 26 58 140 663 46 83
TL
T
HF
E
220 305 352 412 – 247 385 – – 275 338 400 335 356 396
135 145 213 213 – 150 235 – – 169 202 221 192 206 230
25 21 26 28 – 20 29 – – 21 24 26 27 25 26
23 20 23 27 – 12 17 – – 21 24 29 24 26 27
ZB
LIB
NB
CBL
PL
PB
MTL
LM
M1-3
M4
– 20.7 – 21.15 – 23.54 20.58 24.24 17.24 23.03 23.38 – – 21.29
– – 7 – 7.61 – 6.16 6.16 7.43 6.21 7.16 8.35 – – 6.87
– – – – 5.38 – 4.78 4.08 5.46 2.85 3.76 5.67 – – 4.09
– – 37 – 37.42 – 40.01 34.51 41.63 31.02 40.7 40.24 – – 36.73
– – 20.5 – 20.96 – 21.02 18.91 22.84 17.25 22.85 22.44 – – 20.59
– – 12.2 – 13.94 – 13.57 13.13 14.12 11.47 14.14 14.21 – – 13.6
– – 15.5 – 16.86 – 16.49 15.27 17.69 12.47 17.6 17.36 – – 16.68
– – 7.7 – 8.85 – 8.39 8.54 9.05 – 9.24 9.21 – – 9.12
– – 6.6 – 7.66 – 7.13 7.29 7.74 7.23 7.91 7.85 – – 7.54
– – – – 2.83 – 2.84 2.8 2.85 – 3.07 3.13 – – 2.79
measurements and characteristics (Tate 1931; Fig. 2; see above). New locality records fill the gap between previously known localities, showing that this species is widely distributed throughout the eastern forested portion of Paraguay (Fig. 2). In addition, our molecular data, including the first haplotypes from near the type locality (“Villa Rica, Paraguay”; Tate 1931: 1), suggest that only one form of Marmosa (Micoureus) is found and widely dispersed in eastern Paraguay, northeastern Argentina, and southeastern Brazil.
Discussion The phylogenetic analysis shows that the two main lineages of Micoureus are present in Paraguay. One of this is represented by M. (M.) paraguayana that inhabits the forested areas of eastern Paraguay. Meanwhile, the other main lineage is represented by M. (M.) constantiae, which is distributed in open areas of both Chaco and the Cerrado in eastern Paraguay. This finding highlights the diversity of Paraguay not just at the species level but also phylogenetically. This is important given that the mammalian fauna of Paraguay is one of the least known in South America (Pine 1982; Myers et al. 2002) even the long history of reports about these vertebrates (de Azara 1801, 1802; Rengger 1830; Bertoni 1914). This scenario is reflected in several recent additions to the known mammal fauna of Paraguay, which include a marsupial (de la Sancha et al. 2007), a bat (Stevens et al. 2010), several rodents including a new family (D‘Elía et al. 2008; Percequillo et al. 2008; de la Sancha et al. 2009a, 2011), an armadillo (Smith et al., in press), and even an exotic lagomorph (de la Sancha et al. 2009b). Additional field work will aid in improving sample sizes as well as providing much needed taxonomic, ecological, and distributional data about these groups. This is especially urgent in bioregions like the Interior Atlantic Forest which are quickly disappearing (Huang et al. 2007, 2009). Relationships among species of Micoureus are fully resolved (Fig. 1). Interestingly, the most basal dichotomy of the Micoureus clade does not delimit cis- and trans-Andean (sensu Haffer 1967) reciprocally monophyletic groups. M. (M.) alstoni the single transAndean species included in the analysis is well nested in the Micoureus clade; i.e., cis-Andean species form a paraphyletic group with respect to the trans-Andean species (Fig. 4). This result suggests that the colonization of the western (trans) side of the Andes would be a relatively late event in the biogeographic history of Micoureus. In this respect, is of much interest to include in the phylogenetic analysis representatives of M. (M.) phaea, the other trans-Andean species of the subgenus (Fig. 4), to clarify if it crossed the Andes on a single event or if the western side of the Andes was colonized more than once by Micoureus.
Our sampling of paraguayana includes 23 haplotypes, which allows for a preliminary picture of the geographic structure of the genetic variation of this species. The species has genealogical structure but it is not geographically structured (Fig. 1). These results support Gardner and Creighton (2008) whom do not recognize subspecies within paraguayana. Paraguayan haplotypes are well nested in the paraguayana clade rendering the mostly coastal Brazilian sample paraphyletic to Paraguayan variants. A similar pattern was found in the analysis of a small sample of Akodon paranaensis (D‘Elía et al., 2008). In light of this result we suggest that M. (M.) paraguayana would have colonized eastern Paraguay from a source in the Atlantic coastal region. A similar demographic scenario is being suggested for the sigmodontine mice Akodon montensis in association with Atlantic Forest dynamics during the Pleistocene (Valdez and D‘Elía, in prep.; see also Carnaval and Moritz, 2008). Finally, the genealogical analysis shows that additional taxonomic work is needed to clarify the number of distinct biological units, either species or subspecies, within Micoureus. For example, M. (M.) demerarae was not recovered as monophyletic, given that one haplotype (GenBank accession number U34673) assigned to demerarae appears as sister to alstoni. A similar result was already obtained by Dias et al. (2010) with a reduced taxonomic sampling. Haplotype U34673 is highly divergent from all others studied (9.5% respect the alstoni clade; 10.3% respect to the demerarae clade), raising the possibility that the specimen from which it was recovered represents a distinct species of those studied here. Since we have not had the opportunity to study the voucher, we will not proceed further on this issue. In addition to the issue raised by the position of haplotype U34673, results show that M. (M.) regina and M. (M.) demerarae present deep phylogeographic structure. M. (M.) regina shows two strongly supported allopatric clades (Fig. 1) that differ on average by 2.7%. However, sampling is scarce precluding the evaluation of the congruence between this phylogeographic break and the subpecies recognized by Gardner and Creighton (2008). Three main allopatric clades were found for M. (M.) demerarae that, on average, differ among them by 5.0% (Fig. 4b). One clade (1; intraclade variation: 1.9%) distributes in northern Brazil (Amazonas), southern Venezuela, Guyana and French Guiana; clade 1 in turns divides into two subclades: 1a in northern Brazil (Amazonas), and southern Venezuela and 1b in Guyana and French Guiana. The second main clade (2; intraclade variation: 2.5%), sister to clade 1, distributes in central and eastern Brazil (Bahia, Mato Groso, and Tocantins); similarly clade 2 is composed of three subclades that are geographically segregated in the longitudinal dimension. The third main clade (3; intraclade variation: 1.7%) of M. (M.) demerarae, sister to the clade formed by clades 1 and 2, distributes in
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Fig. 4. Distribution of the currently recognized species of Micoureus taken from Gardner and Creighton (2008) and modified according the results of the present study. Localities from were specimens were taken for the phylogenetic analysis are shown; locality numbers are those used in Fig. 1 (see also main text and Supplementary material). (A) Phylogenetic relationships among species of Micoureus as recovered in the cytochrome b gene sequence analysis (see text and Fig. 1 for reference numbers). (B) Phylogenetic relationships among the phylogeographic units found within M. (M.) demerarae. Type localities of the five currently recognized subspecies of M. (M.) demerarae are indicated with gray circles, while those of the two taxa currently considered synonyms of recognized subspecies are indicated with gray circles with a black cross. Taxonomy follows Gardner and Creighton (2008).
eastern Peru and central Brazil (Mato Groso). Analyzing other data sets Costa (2003) and Patton and Costa (2003) found similar results. Even when, as those of Costa (2003) and Patton and Costa (2003), our sampling is geographically partial and does not include any topogenetype (sensu Chakrabarty, 2010), it is clear that the phylogeographic pattern of M. (M.) demerarae is not fully concordant with the subspecific arrangement recognized by Gardner and Creighton (2008) in which five subspecies are considered. For example, as already mentioned by Gardner and Creighton (2008) the phylogeographic unit of Peru and southwestern Brazil (clade 3) apparently lacks a name. However, we note that the type locality of dominus Thomas, 1920, a name currently assigned to populations in eastern
Brazil (i.e., clade 2), is geographically closer to sampled populations of the northern (1) and western (3) clades than to those of clade 2. Therefore, further geographic sampling may show that dominus may in fact apply to the western phylogeographic unit or the northern clade (in which case would be a synomyn of nominotypical subspecies) and not to the eastern one. Then, if proved that dominus applies to either clade 1 or 3, limae Thomas, 1920, a form currently considered a synomyn of dominus and with type locality laying close to the geographic distributional area of the eastern clade (2), may be the correct name to apply to it. The important issue going beyond these taxonomic scenarios and the one that we want to highlight here is that these results, once again, reinforce
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the long standing claim that further basic research involving field collection of additional specimen series and museum based work is still much needed. Acknowledgements We thank Sharon Jansa and Jim Patton whom generously provided unpublished data and Ronald Pine, Alfred Gardner, Shayna Harris, and an anonymous reviewer for comments and revisions of this manuscript. The assistance of Robert Baker and Heath Garder (TTU), Bruce Patterson (FMNH), Phil Myers (UMMZ), Joe Cook and Jon Dunnum (MSB) and the staff at the Museo Nacional de Historia Natural del Paraguay is greatly appreciated. We thank Tom Husband and the Conservation Genetics Laboratoy at University of Rhode Island (URI) where several sequences were generated. This research is based in part upon work conducted using the Rhode Island Genomics and Sequencing Center which is supported in part by the National Science Foundation (MRI Grant DBI-0215393 and EPSCoR Awards 0554548 & 1004057), the US Department of Agriculture (Grants 2002-34438-12688, 2003-34438-13111 and 2008-34438-19246), and the URI. Financial support to NDLS was provided by American Philosophical Society (through the Lewis and Clark Exploration Fund), the Marshall Field Collection Fund of the Field Museum of Natural History in Chicago, the TTU Association of Biologists (TTUAB) mini-grant, the TTU Graduate School, for a AT&T/McNair Fellowship, assistantships from the Department of Biological Sciences (TTU), the Mary Rice Foundation (special thanks to Mrs. Mary Rice), the International Institute for Education (via a Fulbright Scholarship), a Michelle Knapp Memorial Scholarship (TTU), and a Knox Jones Award (TTU). FONDECYT 1110737 and MECESUP AUS0805 provided support to GD. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mambio.2011.10.003. References Anderson, S., 1997. Mammals of Bolivia: taxonomy and distribution. Bull. Am. Mus. Nat. Hist. 231, 1–652. de Azara, F., 1801. Essais sur lˇıhistoire naturelle des quadrupèdes de la Province du Paraguay. Tome Second. Imprière C. Pougens, Paris. de Azara, F., 1802. Apuntamientos para la historia natural de los quadrúpedos del Paraguay y Río de La Plata. Tomo segundo. Imprenta de la Viuda de Ibarra, Madrid. Bertoni, A.de W., 1914. Fauna paraguaya: catálogos sistemáticos de los vertebrados del Paraguay: peces, batracios, reptiles, aves, y mamíferos conocidos hasta 1913 Asunción: Establecimiento Gráfico M. Brossa. Descripción física y económica del Paraguay. Numeración Novenal 59, 1–86. Brown, E.B., 2004. Atlas of New World marsupials. Fieldiana Zool. 102, 1–308. Carnaval, A.C., Moritz, C., 2008. Historical climate modelling predicts patterns of current biodiversity in the Brazilian Atlantic forest. J. Biogeogr. 35, 1187–1201. Chakrabarty, P., 2010. Genetypes: a concept to help integrate molecular phylogenetics and taxonomy. Zootaxa 2632, 67–68. Contreras, J.P., 1984. Notas sobre el género Marmosa en el noreste Argentino (Marsupialia: Didelphidae). Hist. Nat. 4, 11–12. Costa, L.P., 2003. The historical bridge between the Amazon and Atlantic Forest of Brazil: a study of molecular phylogeography with small mammals. J. Biogeogr. 30, 71–86. de la Sancha, N.U., D’Elía, G., Tribe, C.J., Perez, P.E., Valdez, L., Pine, R.H., 2011. Rhipidomys (Rodentia, Cricetidae) from Paraguay: noteworthy new records and identity of the Paraguayan species. Mammalia 75, 269–276. de la Sancha, N.U., Mantilla-Meluk, H., Ramirez, F., Perez, P., Mujica, N., Troche, A., Giménez, M., 2009b. Notes on geographic distribution: Mammalia, Lagomorpha, Leporidae, Lepus europaeus, Pallas, 1778: distribution extension, first confirmed record for Paraguay. Check List 5, 428–432. de la Sancha, N., D‘Elía, G., Netto, F., Perez, P., Salazar-Bravo, J., 2009a. Discovery of Juliomys (Rodentia, Sigmodontinae) in Paraguay, a new genus of Sigmodontinae for the country’s Atlantic Forest. Mammalia 73, 162–167. de la Sancha, N., Solari, S., Owen, R.D., 2007. First records of Monodelphis kunsi Pine (Didelphimorphia, Didelphidae) from Paraguay, with an evaluation of its distribution. Mastozool. Neotrop. 14, 241–247.
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