Outstanding diversity and microendemism in a clade of rare Atlantic Forest montane frogs

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Highlights

  • Frogs in the I. holtiI. lactea species complex inhabit montane Atlantic Forest.

  • We delimited nine genetic lineages, most of which show very narrow ranges.

  • Relationships of lineages in higher elevation were poorly resolved.

  • Past ranges and phylogenetic uncertainty suggest climate driven vicariance.

  • We suggest reassessment of taxonomy and conservation statuses in this species complex.

Abstract

Distributed across topographically complex landscapes that vary from lowland to high elevation, the Atlantic Forest harbors one of the richest biotas worldwide. Atlantic Forest amphibians are particularly speciose, taxonomic accounts are rising and the group is used as model for biogeographic inference. Past climate-related habitat fragmentation is often invoked to explain diversification, with montane taxa expected to become more widespread during glacial times and restrained at interglacials. In this study we investigate diversification in Ischnocnema lactea and I. holti (Anura: Brachycephalidae), two rare frog species inhabiting Atlantic Forest montane regions in Southeastern Brazil. Previous phylogenetic accounts have suggested uncertain limits between these two sister species. We assembled a multilocus DNA dataset, delimited lineages in this clade, and used ecological niche modeling to explore past and future putative ranges. Assignment analyses and traditional and coalescent phylogenetic methods confirmed the existence of a species complex of Miocene origin comprising nine lineages, most of which show very narrow ranges. Lineages were fully supported as species based in coalescent species delimitation, but the phylogenetic relationships among lineages in higher elevation were unresolved. Models of past ranges suggest extensive suitable areas at the last glacial maximum which, along with phylogenetic uncertainty, are consistent with a hypothesis that climate driven vicariance at higher elevation areas resulted in hard polytomies. Species distribution models under future climates suggest narrower ranges of the lineages relative to now, but no species are currently considered endangered. Overall, our results argue in favor for the reassessment of the taxonomic and conservation status of the I. holtiI. lactea species complex.

Introduction

Mountains affect the origin and maintenance of biodiversity directly and indirectly, and are often high biodiverse areas themselves (Hoorn et al., 2013). Montane systems in the tropics sustain even larger number of species and striking degrees of endemism at higher elevations (Cadena et al., 2012; Merckx et al., 2015). Mountain taxa diversification is often explained by neutral and allopatric models: populations become isolated in mountaintops and differentiate after enduring intense genetic drift (e.g. Firkowski et al., 2016), albeit non-neutral and sympatric diversification across elevation gradients have also been explored (e.g. Patton and Smith, 1992).

The Brazilian Atlantic Forest is a hyperdiverse biome distributed across the tropical and subtropical Atlantic margin of the South American continent. In eastern Brazil, topography is especially complex and landscapes vary in elevation from sea level to nearly 3,000 m above sea level (Pico da Bandeira, 2892 m a.s.l.). Diversification hypotheses invoke isolation of populations following Plio-Pleistocene climate-related habitat fragmentation (Carnaval et al., 2009) or populations being bounded by geographic barriers (Thome et al., 2014). In this context, whereas the complex relief is permeated by neotectonic faults and lineaments (Saadi et al., 2002), a preference for the cold would cause montane taxa to respond to past climate change in a particular way, becoming more widespread during glacial times and restrained to mountaintops at interglacials (Amaro et al., 2012, Carnaval et al., 2014, Rodrigues et al., 2009).

Atlantic Forest amphibians constitute one of the most diverse vertebrate groups globally; a recent account listing an impressive number of 625 species likely represents an underestimation as new taxa are frequently described (Rossa-Feres et al., 2017). Because of particular life history traits that enhance phylogeographic signal retention, such as low vagility and habitat specificity (Avise, 2000), Atlantic Forest amphibians have been used as models to elucidate the origins of the biome's biodiversity, attributed to a combination of distinct evolutionary mechanisms and deep histories (Turchetto-Zolet et al., 2013). Particularly for amphibians, diversity is sustained by a combination of large altitudinal and latitudinal ranges with a complex relief creating moisture gradients and climatic conditions that vary at local scale (da Silva et al., 2012, Haddad and Prado, 2005).

One of the most emblematic anuran families in the Atlantic Forest is the Brachycephalidae, comprising ground-dwelling, direct-developing frogs in the genera Brachycephalus and Ischnocnema (Hedges et al., 2008). Both show high species diversity and extreme endemism levels. About half of the Brachycephalidae species were described in the last decade (Frost, 2019), and the availability of molecular data has played a fundamental role in solving phylogenetic uncertainty within complexes of cryptic species. A relevant aspect of the ecology of the family is the preference for montane habitats and very restricted ranges; often, its species span a single mountaintop. This trait is prevalent in Brachycephalus but less frequently reported for Ischnocnema, although recent molecular studies revealed that several Ischnocnema species correspond, in fact, to complexes of multiple, highly divergent lineages with restricted distributions (e.g. Gehara et al., 2017, Taucce et al., 2018a), including cases of microendemism (I. guenteri, Gehara et al., 2013).

Ischnocnema currently comprises 38 described species distributed in five species series, as supported by recent phylogenetic studies; they are the I. guentheri, I. lactea, I. parva, I. venancioi, and I. verrucosa species series (Canedo and Haddad, 2012, Taucce et al., 2018b). The Ischnocnema lactea species series is at the base of the Ischnocnema tree and comprises 11 species (Canedo and Haddad, 2012, Taucce et al., 2019). Within this series, a particular clade containing I. lactea (Miranda-Ribeiro, 1923) and I. holti (Cochran, 1948) was highlighted as a possible species complex, given that I. holti was recovered paraphyletic in relation to I. lactea (Canedo and Haddad, 2012, Taucce et al., 2018a, Taucce et al., 2018b). The two species are associated to montane Atlantic Forest physiognomies in Southeastern Brazil, being notably rare in the field and poorly represented in museum collections. Ischnocnema lactea inhabits the Serra do Mar mountain range at moderate elevations (700–1120 m a.s.l., Silva-Soares et al., 2018), and was recently redescribed by Silva-Soares et al. (2018). Ischnocnema holti was redescribed by Targino and de Carvalho-e-Silva (2008), who restricted its occurrence to a single high elevation site in the Serra da Mantiqueira mountain range (Itamonte, 2000–2400 m a.s.l.). Costa et al. (2008) expanded this geographic distribution to Serra dos Órgãos, and specimens of dubious identification (morphologically similar to I. holti) were also found in other high elevation areas of southeastern Brazil (see Table 1, Fig. 1). Little is known about natural history of the populations assigned to the two species names, with individuals of I. lactea being reported to reproduce during cold fronts in the winter season, under relatively low temperatures (Silva-Soares et al., 2018).

In this study, we prospect tissue samples from major museum collections in southeastern Brazil to access the genetic diversity of the rare I. holtiI. lactea species complex. We delimit lineages by combining traditional frequency and tree-based methods with coalescent methods under a multilocus approach. We use a molecular clock to define a time window for diversification in the complex, and explore aspects of its evolutionary history by contrasting lineage distributions to maps of geographic barriers and past putative ranges obtained with ecological niche modeling (ENM). Finally, we project the I. holtiI. lactea species complex climatic niche to the future and discuss its conservation under a global climate change scenario.

Section snippets

Taxon and gene sampling

We gathered tissue samples from vouchers previously identified as Ischnocnema holti, I. cf. holti, I. aff. holti, I. lactea, I. cf. lactea, I. aff. lactea, I. gr. lactea, Ischnocnema sp. (gr. lactea), and Ischnocnema sp., deposited in six main tissue collections in Brazil. This strategy resulted in 37 tissue samples from 18 localities (Fig. 1, Table S1). We also sampled five species to be used as outgroups in phylogenetic analyses, following results in Canedo and Haddad, 2012, Taucce et al.,

Genetic structure and phylogenetic inferences

Structure analyses suggest the existence of seven unique genetic clusters within the I. holtiI. lactea species clade. The plot with mean LnP(K) across K values shows an inflexion point at K = 7, with similar mean LnP(K) values above that. The Evanno criterion ΔK peaks twice, at K = 2 and K = 7 (Fig. 2A), indicating hierarchical genetic structure: population limits at K = 2 are also recovered at K = 7 (Fig. 2, Table 1). At K = 2 all samples are attributed with high q values, being clustered

Discussion

In this study we combined assignment analyses with phylogenetic inference to prospect lineages within the I. holtiI. lactea species complex, validated those lineages under the multispecies coalescent model, and used ENM to explore its past and future putative range. We detected nine lineages that were fully supported as species, but their phylogenetic relationships were not completely resolved. Extensive suitable areas at the LGM, along with phylogenetic uncertainty, are consistent with

CRediT authorship contribution statement

Maria Tereza C. Thomé: Writing - original draft, Formal analysis. Mariana L. Lyra: Data curation, Writing - review & editing. Priscila Lemes: Formal analysis, Writing - review & editing. Laryssa S. Teixeira: Data curation. Ana Carolina Carnaval: Writing - review & editing. Célio F.B. Haddad: Resources, Funding acquisition, Data curation, Writing - review & editing. Clarissa Canedo: Conceptualization, Supervision, Writing - review & editing.

Acknowledgments

We thank José P. Pombal Jr. (Museu Nacional, Rio de Janeiro - MNRJ), Paulo Garcia (Coleção de Anfíbios do Centro de Coleções Taxonômicas da Universidade Federal de Minas Gerais - UFMG), Miguel Rodrigues (Coleção MTR do Instituto de Biociências da Universidade de São Paulo - MTR), Marcia dos Reis Gomes (Coleção de Anfíbios do Laboratório de Anfíbios e Répteis do Departamento de Zoologia, Instituto de Biologia, Universidade Federal do Rio de Janeiro - ZUFRJ) and Taran Grant (Coleção de Anfíbios

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