The importance of museum specimens for the study of Neotropical tree squirrels
All samples used on this study were gathered from specimens deposited in scientific collections. The inclusion of historical samples was crucial in the detection of several taxonomic issues reported here. Almost a third of our samples were obtained from dry museum specimens, collected between 1893 and 2010, and housed mostly in two North American museums (AMNH and USNM). We were successful in obtaining at least 20% of the mitogenome for about 70% of the historical samples, which allowed us to include 18 nominal taxa in the Sciurini phylogeny for which ethanol-preserved tissues were not available. Moreover, two of the main groups recognized within Sciurini (B and H) were exclusively represented by historical samples.
Our success in obtaining mtDNA data from historical samples is twofold: i) it could be partially attributed to the sequencing method employed. Next-generation sequencing techniques (e.g. shotgun sequencing, targeted sequencing via hybridization-based captures or via restriction enzyme-based enrichment [28]) are highly advantageous for obtaining large-scale genomic data from historical samples in comparison with traditional sequencing methods (e.g. Sanger sequencing), as they are very efficient in sequencing short fragments of DNA [14], which are expectedly abundant in old museum samples; ii) it could also be a consequence of our sampling strategy. We prioritized obtaining fragments of muscular tissue adhered to skulls, which had been show by previous studies [12] and confirmed here, to yield higher concentrations and longer fragments of DNA than samples obtained from skins clips.
The use of historical samples in molecular studies has increased substantially in the last two decades (see review in [14, 29]) and is helping to reveal hidden diversity, to unveil puzzling phylogenetic relationships, and to place rare and elusive mammal species/lineages in a phylogenetic context [16, 30–32], including in squirrels [33]. For Neotropical mammal groups, the study of historical museum samples using high-throughput sequencing to address phylogenetic and taxonomic questions is growing, becoming more feasible, and holding lots of promise [34, 35].
When investigating how different aspects of historical samples of Sciurini might influence the success of mitogenome recovery, our most robust result was that sample age (the year in which the specimen was collected) does not affect the completeness of mitogenomes obtained (Figure 1). Previous studies that compared age of source sample with other metrics of mitochondrial DNA recovery (e.g. copy number) also found no relatedness between those two factors [12, 15, 36]. Together, those finds highlight the potential of old museum specimens— including holotypes and other taxonomically important material— for phylogenetic and evolutionary inferences. Future studies on Neotropical mammals could benefit from the large series of specimens collected in South America during the first decades of the 20th century (e.g. by A. Garbe, see [37]; and by the Olalla family, see [38]). These valuable and irreplaceable specimens—many of those from localities that were long-ago transformed into human modified landscapes—will allow the reconstruction of comprehensive phylogenetic hypotheses for taxonomic groups for which samples of fresh, frozen, or ethanol-preserved tissue are absent, scarce, and/or difficult to obtain.
Despite the immense value of historical material, as aforementioned, we advocate that this type of sample should be used as a complement to traditional ethanol-preserved samples. As documented by our and previous studies [12], modern samples result in higher sequencing success, and they have the great advantage of not being destructive, in any sense, to the morphological vouchers. Therefore, whenever possible, they should be preferred as a primary source of genetic data. In this sense, contemporary field sampling using diverse collection techniques is crucial to increasing the representativeness and value of our repositories of biodiversity, as defended by Voss and Emmons [9]. The continuity of field expeditions, especially to remote and unsampled regions to collect new specimens, will certainly result in the discovery of many unknown species, which is imperative to uncover the still hidden biodiversity of the richest areas of the globe, including the Neotropics [39]. Ultimately, while historical specimens are a critical and essential resource, they should not obscure the potential value of obtaining additional specimens for science in the wild, which are paramount not only for taxonomic and phylogenetic refinement, but also for documenting ongoing ecological and evolutionary changes and promoting biodiversity conservation.
Missing data versus missing taxa
The discussion regarding how much missing data (and their effects) should be allowed in phylogenetic inferences has gained considerable attention, especially after the dissemination of next-generation sequencing methods (e.g. [40–43]). It was previously shown that missing data could obscure phylogenetic relationships and promote negative impacts on the phylogenies (e.g. [44]). Subsequent authors (e.g. [41]) have shown that including as many loci as possible in the phylogenetic analyses is benefical even with large amounts of missing data because this would increase the sampling of distinct regions across the genome. Streicher et al. [42] emphasized that the optimal approach in terms of amount of missing data incorporated without losing the accuracy of the inference depends on the dataset and the phylogenetic method employed. After exploring our data by comparing the performance of matrices with alternative sampling strategies, we observed that the addition of samples with a limited amount of missing data did not impact the estimated relationships nor cause significant change to the nodal support of the inferred phylogenies.
Mitochondrial phylogeny and taxonomic arrangements proposed for Sciurini
The comparison of our results with proposed taxonomic arrangements for Sciurini ([2, 18, 21] and [1] which is identical to [20, 22]) illustrates that none of the generic arrangements fully corresponds to the phylogenetic structure recovered (Figure 5). Most currently recognized genera (by [1, 2]) are not recovered as monophyletic by our analyses of genetic data. Allen [18] suggested the greatest diversity of genera, and his hypothesis seems to be the one that best fits our results, especially regarding the Nearctic taxa.
The delimitation of the taxa at the genus-group level has not received as much attention as species delimitation [45, 46]. Recently published generic arrangements for South American rodents, which included the description of new genera [47–49], have provided a solid diagnosis for the new taxa by consistently testing phylogenetic hypotheses and employing reciprocal monophyly as a primary criteria. Their phylogenetic analyses were complemented with robust and consistent morphologic analyses and they then applied total evidence analysis or conducted a posteriori comparison to support taxon diagnosis.
Here we use the phylogenetic information provided by a taxonomically robust mitogenome dataset to suggest a tentative classification at the genus level for Sciurini (Figure 6). In our arrangement, we recognize reciprocal monophyletic entities as taxa at the genus-group level, and most of those entities correspond to groups A to L as recovered by our analyses; the only exception is the recognition of three reciprocally monophyletic genera within Group G, based on previous classificatory arrangements [1, 2, 18, 21]. We attribute to them the appropriate available names following the criteria established by the International Commission on Zoological Nomenclature (ICZN). Therefore, we favor the principles of priority and stability in such tentative nomenclatural acts, employing whenever possible the generic names proposed by Allen in 1915 [18], the first reviewer and author of several names of the genus-group valid and available for this radiation of squirrels.
For the sake of consistency with current generic nomenclatural acts (see above), our limited morphologic dataset (number of pairs of mammae and of upper premolars) precludes us from providing formal diagnosis and description for a presumptive taxon, a goal that is beyond the scope of this contribution. Thus, for the lineage of the genus-group level with no available name, we apply the genus name that was historically employed for it, presenting this name between quotation marks.
Given the limitations and shortfalls of our data, which are solely based on mitochondrial DNA, we do not presume this updated generic arrangement the definitive scheme, but we intend to offer a working hypothesis that can be tested and formalized by further studies, as additional data become available. Careful taxonomic assessments with the inclusion of several lines of evidence, such as phenotypic information from sequenced and type material, are indispensable for this and other taxonomic issues of Sciurini to be properly addressed.
The first two major groups within Sciurini (A and B) compose the genera Tamiasciurus Trouessart, 1880 (including T. douglasii and T. hudsonicus) and Rheithrosciurus Gray, 1867 (including R. macrotis) as monophyletic groups, and we suggest the application of these names for the Groups A and B, respectively. The genus Sciurus, as broadly recognized in the past century—including Eurasian, Nearctic and Neotropical species (e.g. [1, 20–22])—, is not monophyletic. This result has been also recovered in previous phylogenetic inferences with fewer species [7, 50–52] and is strongly supported by our analyses with denser taxon sampling. Sciurus can be restricted to the Eurasian clade, Group C, since it includes vulgaris Linnaeus, 1758, the type species of Sciurus Linnaeus, 1758. Other species included in the restricted concept of this genus are S. anomalus and S. lis. The remaining North American species are arranged in Groups D, E and F, for which four generic names are available, Hesperosciurus, Otosciurus, Neosciurus and Parasciurus. For Group D, two generic names were coined as subgenera by Nelson in 1899, Otosciurus for Sciurus aberti and Hesperosciurus for S. griseus. Here we conservatively assign the oldest available genus-group name, Hesperosciurus, as a genus including H. aberti and H. griseus. Regarding Group E, the genus name Parasciurus Trouessart, 1880 is the only available one, and its type species is niger Linnaeus, 1758; therefore, we suggest the application of this name for this clade, composed of P. nayaritensis, P. arizonensis, P. alleni and P. oculatus, along with the type species. Finally, for Group F the only available name is Neosciurus, described by Trouessart, 1880 for carolinensis Gmelin, 1788, and this is the name that we tentatively apply to this lineage.
Group G, which comprises most Central American taxa, might be the most taxonomically conflicting group as it contains the type species of many genera, including alfari (type of Microsciurus J. A. Allen, 1895), brochus (type of Syntheosciurus Bangs, 1902) deppei (type of Baiosciurus Nelson, 1899) and aureogaster (the senior synonym of Sciurus hypopyrrhus Wagler, 1831, type species of Echinosciurus Trouessart, 1880) (Figure 5). Allen [18] recognized five genera for the eight species or species-complex in this group. Subsequent authors recognized fewer genera, but none suggested a unique genus to contain those species. It is noteworthy that Moore [21] was the only author to anticipate a close relationship between brochus and granatensis, suggesting these taxa be placed under the genus Syntheosciurus. We partially follow the arrangement proposed by Allen [18] and Moore [21], and we suggest that the genus name Microsciurus should be applied for the clade formed by alfari and “species 1”; the name Syntheosciurus must be attributed to the group formed by brochus and granatensis (if Vivo and Carmignotto are correct [see below, on the discussion of the name of clade H] and granatensis is the type species of Notosciurus, this genus name is a junior synonym of Syntheosciurus); and we advocate the adoption of the name Echinosciurus, as the oldest available one, for the group formed by aureogaster, colliaei, deppei, yucatanensis and variegatoides.
Group H, composed of northwestern South American forms, includes five species traditionally allocated to the genus Microsciurus J. A. Allen, 1895 (mimulus, similis, otinus, boquetensis, and isthmius). However, the type species of the genus Microsciurus (alfari J. A. Allen, 1895), as demonstrated above, is nested within Group G and, therefore, the name Microsciurus cannot be applied to Group H. The other species recovered in this clade, pucheranii Fitzinger, 1867, is a controversial taxon. Allen [18] described the genus Leptosciurus and considered pucheranii as its type species. Moore [21] placed pucheranii in Microsciurus, and he was the only author to suggest a close relationship between this taxon and the small-sized species from the highlands of northwestern South America. Most other authors have allocated pucheranii to Sciurus (e.g. [1, 19, 20]), but Vivo and Carmignotto [2] included pucheranii and granatensis under the genus Notosciurus Allen, 1914. Their decision was based on the fact that: i) their concept of granatensis included chysuros Pucheran, 1845 as a subspecies (N. g. chrysuros) and soederstroemi Stone, 1914 as a junior-synonym of this subspecies; ii) Vivo and Carmignotto [2] followed Hershkovitz [53] who identified N. rhoadsi Allen, 1914 (the type species of Notosciurus) as a young specimen of soederstroemi. Therefore, for these authors, the name Notosciurus would be applied to granatensis (via its synonymy with soederstromi) and pucheranii (for their morphological similarity), and would have priority over the name Leptosciurus. However, our results did not recover granatensis and pucheranii as closely related taxa. Instead, granatensis was recovered in Group G, sister to Syntheosciurus brochus. Therefore, Leptosciurus seems to be the only available name for Group H, and we suggest the application of this name to the six species there nested.
Group I includes stramineus (type species of Simosciurus Allen, 1915) along with nebouxii. Vivo and Carmignotto [2] followed Allen [18] considering Simosciurus a valid genus, and we recover it as monophyletic based on mitogenomic data. Since Simosciurus is the only available name for this clade and it includes the type species of this genus, we believe it is the appropriate name for Group I. Our analyses also support the monophyly of the genus Guerlinguetus Gray, 1821, as recognized by both [2, 18], represented in our analyses by Group J. Guerlinguetus has been consistently employed for parts of this particular group of species, as full genus or subgenus, by several authors.
Described species recovered within Group K have been assigned to the genus Microsciurus by all authors. The unnamed lineage recovered within this Group (“species 2”) was also referred to the genus Microsciurus by [54, 55], but it has been referred to the genus Syntheosciurus by Vivo and Carmignotto [2]. Our data do not recover taxa of this group as closely related to the type species of Microsciurus or Syntheosciurus. Moreover, as the valid species in this group (flaviventer and sabanillae) were both described in genera currently occupied (Macroxus and Microsciurus, respectively), there seems to be no generic name available for Group K. Until more consistent morphologic dataset is available to allow a formal nomenclatural designation, we provisionally use the name “Microsciurus” for this clade (see Patton et al., 2015, for “Handleyomys”), as this was the name historically assigned to these species. An alternative measure would be to apply the genus name of the sister group (L) to this lineage, but we do not recommend this option as this would introduce more taxonomic confusion and instability.
Finally, Group L clustered species allocated in distinct genera according to [2, 18], or from a single but not monophyletic genus of Moore [21] and Thorington et al. [1]. At least five generic names have been applied to those species: Notosciurus Allen, 1914, Leptosciurus Allen, 1915, Mesosciurus Allen, 1915, Hadrosciurus Allen, 1915, and Urosciurus Allen, 1915. However, only Hadrosciurus and Urosciurus are possibly vacant here, and the correct assignment must be carefully evaluated in a comprehensive taxonomic study that includes a meticulous nomenclatural investigation for this group. However, in order to propose a tentative nomenclatural definition, as we have done for previous clades, we tentatively apply the name Hadrosciurus Allen, 1915, whose type species is flammifer Thomas, 1904, considered by Vivo and Carmignotto [2] as a junior-synonym of igniventris Wagner, 1842. This name was also advocated by Vivo and Carmignotto [2].
Comments on species recognition and novelties
In this study, we sampled across the geographic ranges of several widespread taxa and, therefore, we were able to test the genetic integrity of currently recognized species of tree squirrels, especially those from South America. In contrast to our generic level analyses, most recognized species are highly supported as monophyletic groups in our analyses of mitochondrial genome data. Regarding Palearctic and Nearctic taxa, our sampling was remarkably inferior to the sampling for Neotropical taxa, with many species represented by as few as one or two individuals. As expected, all those species with more than one individual exhibit reciprocal monophyly in our phylogenomic analyses, following the species concepts presented by Thorington et al. [1].
Among the Central American taxa, the two cases of non-reciprocal monophyly were (i) the recovery of a sample identified as richmondi (Nelson, 1898), from Nicaragua, nested within the clade associated with Syntheosciurus granatensis; and (ii) a specimen assigned to venustulus (Goldman, 1912), from Panama, nested within the clade of Microsciurus alfari. Samples from Syntheosciurus granatensis compose two well-structured subclades, one of which includes specimens from the Ecuadorean and Peruvian Andes, Venezuela, and Trinidad and Tobago, and the other includes samples from the coast of Ecuador, Colombia, Nicaragua (referred to as richmondi), and Panama (Group G, Figure 4a). Without the inclusion of additional specimens referred to richmondi and the careful examination of voucher material, we are unable to unveil, at this point, if this is a simple case of misidentification or if this taxon needs taxonomic re- evaluation. Our molecular species delimitation analyses provide distinct resolutions for the samples assigned to granatensis and richmondi, but none of them suggested the sample assigned to richmondi as a distinct species from the specimens of granatensis. Based on our phylogenetic results corroborated by BPP analysis, we recognize a single putative species, Syntheosciurus granatensis, for those samples. Regarding the second case, all samples of Microsciurus alfari, as well as the sample initially identified as venustulus, are from Panama and were suggested as a single species by all species delimitation analyses. Thus, we provisionally do not treat venustulus as a valid taxon until further evaluation with additional specimens.
Across South American lineages, our results indicate that pucheranii sensu [2] forms a non-monophyletic assemblage composed of two phylogenetically distant lineages included in Groups H and L. The concept of pucheranii adopted by Vivo and Carmignotto [2] includes specimens with a disjunct distribution, from the Central Andes of Colombia (assigned to pucheranii pucheranii Fitzinger, 1867) and from Peru and Brazil, Bolivia, and Argentina [assigned to three other subspecies named pucheranii ignitus (Gray, 1867), pucheranii boliviensis (Osgood, 1921), and pucheranii argentinius (Thomas, 1921), respectively]. In our analyses, specimens of pucheranii pucheranii are recovered as part of Group H (Figure 4b)—an Andean Trans-Andean clade composed of taxa from high elevation areas of northwestern South America. For this clade we provisionally apply the name pucheranii Fitzinger, 1867 to the species level, with the combination Leptosciurus pucheranii. Specimens associated with the remaining three subspecies were recovered as a clade nested within Group L (Figure 4c), which includes Cis-Andean lowland taxa. For this lineage we suggest the application of the name ignitus (Gray, 1867), as it has priority over argentinius and boliviensis, with the status of a full species. We did not intend to revalidate or describe new species in this contribution, however, as we were unable to use the current species concepts for the taxa mentioned above, we tentatively suggest this alternative arrangement, which is in accordance with the classification of Thorington et al. [1] at the species-group level; the name we propose is, thus, Hadrosciurus ignitus.
The concepts of Guerlinguetus aestuans and G. brasiliensis adopted by Vivo and Carmignotto [2] are also not monophyletic according to our analyses. Based on the geographic distribution of the samples, the subclades G. aestuans “a” and G. aestuans “b” include specimens associated with Guerlinguetus aestuans. The first subclade is composed of samples from Guyana and Venezuela, and the second of Brazilian samples from the southern bank of the Amazon river, west of the Tapajós river (Figure 5d). The subclade G. aestuans “c” seems to encompass representatives of both aestuans and brasiliensis, since it includes specimens from north of the Amazon river (assigned to aestuans by those authors) and one specimen from Pernambuco, northern Atlantic Forest (assigned to brasiliensis by those authors). We referred to this last subclade as G. aestuans “c” as the great majority of samples within this lineage were previously assigned to G. aestuans and not to G. brasiliensis. The subclade G. brasiliensis is apparently composed of samples assigned exclusively to this nominal taxon, from southeastern Amazonia, eastern and southern Brazil. Therefore, we recognized specimens previously identified as Guerlinguetus aestuans and G. brasiliensis as composing four distinct lineages (see Figure 4b), suggesting hidden diversity along the Amazon basin and implying an independently evolving lineage from the Gran Sabana and Mount Roraima, on the border of Brazil, Venezuela, and Guyana. This result was corroborated by most species delimitation analyses, except for one analysis (GMYC 2) in which Guerlinguetus aestuans “c” and G. brasiliensis were suggested as a unique putative species.
Our phylogenetic results also indicate the existence of three apparently unnamed lineages that might represent species to be described or revalidated, all of which were supported by molecular species delimitation methods. “Species 1” is represented by a specimen from Chocó, Colombia, which was previously identified as Microsciurus mimulus; however, this specimen was recovered as phylogenetically distant from other specimens of M. mimulus from Colombia and Ecuador (all of which clustered within Group H), and exhibited deep genetic divergence from its sister-taxa, M. alfari (see branch lengths on Figure 4a). “Species 2” is represented in our analyses by five specimens from Peru (San Martin, Madre de Dios) and Brazil (Acre). Voucher material of this species, from San Martin, was analyzed by [54, 55]—who referred to it as Microsciurus sp.—and by [2]—who referred to it as Syntheosciurus sp. “Species 3” is represented by three specimens from two Amazonian lowland localities in Loreto (Peru), and is apparently sympatric with Hadrosciurus spadiceus at Rio Galvez, Nuevo San Juan. We did not find previous mention of this putative species in the literature.
Therefore, monophyletic groups representing currently recognized species in addition to the lineages representing putative unnamed taxa composed a set of 43 OTUs that we hypothesize as distinct species of tree squirrels. All South American OTUs were corroborated as unique species by at least two out of the three species delimitation analyses performed, except for one OTU, Leptosciurus boquetensis, which was only supported as a distinct species by BPP. Regarding non-South American taxa, our species delimitation analyses did not fully corroborate our working hypothesis. Discrepant results in the recognition of those species are likely a product of our sampling strategy, densely focused on South American taxa. At least BPP analyses are potentially affected by the number of samples per each presumed species, especially if using a dataset with few loci [56]. GMYC estimates might not be as affected by poorly represented species as BPP [57, 58], but can be strongly influenced by the way that the ultrametric tree is generated, which underprints the analysis [59]. Our results corroborate this assumption, as we found discrepant results suggesting 66 or 39 putative species using ultrametric trees generated with strict and relaxed molecular clocks, respectively.
Several studies have employed molecular species delimitation methods either as a standalone tool or as part of an integrative approach to delimit species [60–62]. Here, we advocate for the use of molecular species delimitation methods along with other sources of evidence, to avoid misleading species delimitation due to theoretical and/or methodological shortfalls (as exemplified above; see also [57–59]). Moreover, in many cases, when delimiting species based on a single-locus dataset, the estimates could be biased by the genealogical history of this locus which may or may not reflect the evolution of the group. As we used an exclusively mitogenomic dataset, we acknowledge that the evidence for pervasive natural selection, uniparental inheritance and the lack of recombination on the mitochondrial genome make it susceptible to evolutionary processes distinct to the nuclear genome [63, 64].
Considering the possible methodological weaknesses mentioned above and the shortfalls of our sampling of taxa and data, we evaluate the results of our molecular species delimitation analyses with special caution in some situations. For example, the genus Tamiasciurus was recently extensively revised through molecular analyses (including mitochondrial and nuclear genes) and ecological niche modeling, with over 250 specimens examined from throughout the distribution of the genus [23]. These authors found evidence for the recognition of T. douglassi and T. hudsonicus as valid species. Our analyses consistently failed to suggest these taxa, represented by a single sample each, as distinct species (see Figure 4a). Another example is that some species delimitation analyses did not recognized Sciurus lis and S. vulgaris (GMYC 2) or Sciurus lis, S. vulgaris and S. anomalus (BPP) as distinct species. These taxa, which are represented in our dataset by only one terminal each, have been consistently recognized as distinct species based on molecular [24, 65] and karyotypical [26] data. They also exhibit consistent morphological differences in the number of pairs of mammae (a trait that seems not to be variable within species of tree squirrels [2]), which is three in S. lis, four in S. vulgaris, and five in S. anomalus (see Figure 6).
These controversial results, especially for Eurasian and North American taxa, lead us to adopt a conservative posture that does not totally reject the hypotheses provided by the species delimitation analyses, but it is also in consonance with current taxonomic proposals based on wider sampling approaches (see examples above). For those cases of inconsistency regarding Eurasian, North American, and Central American taxa, and also for South American lineages where species complexes were suggested by one or two of the species delimitation analyses, subsequent investigations are certainly necessary. A thorough delimitation of species of Sciurini demands additional sampling for several taxa and, possibly, the inclusion of other lines of evidence such as phenotypic and genetic data from independently evolving nuclear DNA loci.
Phylogenetic and biogeographic remarks
Despite the discordances between our mitochondrial phylogenomic hypothesis and the taxonomic arrangements previously proposed for Nearctic and Neotropical tree squirrels, our results are biogeographically coherent, and consistent with most of the results obtained by the few molecular phylogenetic studies published for Sciurini, especially regarding the deepest nodes (major clades) within the tribe. Like Pečnerová and Martínková [66] and Pečnerová et al. [7], we recovered the genus Tamiasciurus as the first lineage to diverge within Sciurini, followed by Rheithrosciurus and Sciurus, although our study is the first to recover strong support for these relationships. Our results also corroborate the sister-taxa relationship between Hesperosciurus griseus and H. aberti found in those previous studies. The Central American clade obtained by [7] is similar in composition to our Group G, despite the different relationships within this group, recovered by us with strong support. In previous studies, the representativeness of South American taxa was very limited, and the relationships among the very few specimens were mostly discordant from our results. One relevant difference is that we recovered the Mexican endemics Parasciurus alleni and P. oculatus clustering with North American species, instead of within a South American clade as in [7].
Concerning the biogeographic pattern, we recovered two Palearctic clades (A and B), four Nearctic (C–F), and six Neotropical—one (G) predominantly composed of Central American with a few South American specimens included (all from Andean or Trans-Andean areas) and five (H–L) composed exclusively of South American taxa and Southern Panama specimens (Figure 4). The distribution of those five predominantly South American clades seems to be defined by the Andean Cordillera. We found two clades occupying Andean and Trans- Andean areas (H and I) and three clades distributed on the Cis-Andean portion of the Continent (Groups J–L). Group H seems largely associated with montane habitats, while Group I is restricted to low elevation coastal areas near the sea-level. Regarding the Cis-Andean groups, Group J is the most widespread, occurring from the extreme east of South America, in the Atlantic Forest, to the Guiana Shield, and throughout the Amazon basin. The sister Groups K and L are largely sympatric and composed mostly by Amazonian lowland dwellers. In Group K, however, two lineages (“Microsciurus” sabanillae and “species 2”) reach mid-elevations on the east side of the Andean cordillera in Ecuador and Peru; and in Group L, one lineage (Hadrosciurus ignitus) is also found in high-altitude localities in Bolivia.
Taxonomic consequence of the use of homoplastic traits in the study of tree squirrels
Historically, all genera proposed for Neotropical species of Sciurini were delimited based exclusively on morphological traits. For example, species of Notosciurus sensu [2] were diagnosed by the presence of three pairs of mammae and one upper premolar; and the genus Microsciurus sensu [1, 2, 18, 21] was defined, among other traits (e.g. small size), by the presence of three pairs of mammae and two upper premolars. Our results, however, indicate that these features are homoplastic, with similar conditions of both characters having evolved multiple times during the evolutionary history of tree squirrels. Morphologic convergence has been detected among several lineages of Sciuridae [67, 68] and, according to our data, seems to be common in both cranial and external traits of Sciurini. Grouping species based primarily on homoplastic characteristics might have led to some of the incongruences that we observe between the taxonomic arrangements and the molecular phylogeny recovered for tree squirrels. For instance, the genus Microsciurus sensu [1, 2, 18, 21] comprises a polyphyletic assemblage that clusters species sharing the same number of premolars and mammae. Phenotypic convergence has been previously detected for cranial traits in species formerly assigned to Microsciurus [7], and the use of homoplastic characters to diagnose this genus (e.g. by [2, 18]) can be claimed to explain the polyphyly of this taxon.