Note on the phylogenetic position of Eptesicus species in the Vespertilionidae family (Chiroptera) using Brazilian bats and RAG2 sequences

The phylogenetic position of the Eptesicus genus in the Vespertilionidae family has been constantly changed after morphology, cytogenetics and molecular studies. For example, the reallocation of some African Eptesicus species from a subgenus of the Pipistrellus genus to the Neoromicia genus (Kearney et al., 2002), and also the reallocation of E. nasutus to the Rhyneptesicus genus (Juste et al., 2013), both indicated uncertainty about the monophyly of this genus. Moreover, New World species of Eptesicus seems to be more closely related to species of Histiotus than they are to Old World species, with Histiotus being considered a subgenus of Eptesicus (Roehrs et al., 2010; Amador et al., 2016).

Therefore, it is clear that the Vespertilionidae family still has phylogenetic issues to be cleared out, specially regarding the positioning of Eptesicus species within the genus and its relation with the Histiotus genus. Hence, the more studies that can be carried out on tribes, subfamilies, genera and related species, the better will be the contributions to shed more light on the understanding of phylogenetic relationships within this family. Thus, in this work we performed a phylogenetic analysis using the DNA obtained from a specimen of Eptesicus furinalis collected in Southern Brazil and also sequences from all other Brazilian species that are available in the GenBank plataform, using the sequence of RAG2 nuclear gene.

Fresh tissue samples of liver, spleen, lung and kidney of Eptesicus furinalis collected in Guarapuava/PR, Brazil (25º 21' 9.40” S 51º 28' 0.30” W), was submitted to DNA extraction using the DNeasy Blood & Tissue (QIAGEN) kit. The other sequences of eleven Brazilian species of the Vespertilionidae family and of Molossus molossus (used as outgroup in the phylogenetic analyses) were obtained from GenBank databank (Table 1). Eptesicus furinalis RAG2 sequences (GenBank accession number: KX961304) were obtained via PCR amplifications, using RAG2F1 and RAG2R1 primers (Baker et al., 2000).

Table 1
Eleven Vespertilionidae species and Molossus molossus (used as outgroup), with GenBank accession number, used in the phylogenetic analysis.

Species	Accession Number	Species	Accession Number	Species	Accession Number
Eptesicus brasiliensis	HM561644	Lasiurus ega	HM561639	Myotis nigricans	GU328088
Eptesicus diminutus	GU328056	Myotis ruber	AM265688	Myotis riparius	GU328089
Histiotus macrotus	HM561648	Myotis albescens	GU328076	Myotis simus	AM265691
Lasiurus cinereus	HM561638	Myotis levis	GU328085	Molossus molossus	AY141017.1

Complementary DNA sequences were assembled for each accession using Geneious® 8.0.2 (Biomatters, 2015). Multiple-sequence alignment was performed using Clustal W (Chenna et al., 2003) followed by manual optimization. Preliminary phylogenetic analyses were conducted to explore the distribution of phylogenetic signal in RAG2 data matrix with and without coded gaps. The resolution and support were affected in substantial way by inclusion of gaps, with gaps treated as fifth base (results shown). Phylogenetic analyses of RAG2 were performed using maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI). The consistency index (CI; Kluge and Farris, 1969) and retention index (RI; Farris, 1989) were calculated. Based on the Akaike information criterion (Posada and Buckley, 2004), the dataset was evaluated for the appropriate model of molecular evolution using ModelTest v.3.7 (Posada and Crandall, 1998). Clade support for both MP and ML phylogenies were assessed with a bootstrap analysis using 1,000 replicates with 100 random addition sequence replicates, and tree bisection and reconnection (TBR) branch swapping as implemented in PAUP* 4.01b10 (Swofford, 2002) using the interface of Geneious® 8.0.2. The BI tree was implemented in MrBayes v.3.1.2 (Ronquist and Huelsenbeck, 2003) using the interface of Geneious® 8.0.2. Four Markov Chain Monte Carlo simulations were run simultaneously and sampled every 100 generations for a total of 1,000,000 generations until the average deviation of split frequencies was well below 0.01.

All three phylogenetic analyses performed in this study (Figure 1), using only Brazilian species of bats, showed Histiotus macrotus as a separated clade from Eptesicus species clade, with high supported nodes. These results were not observed by Giménez et al. (2019), which observed a weak support for this separation using one nuclear and one mitochondrial gene, mostly because of the incongruence of ML with the BI and MP analyses. Moreover, Jones et al. (2002) found that Histiotus is phylogenetically distant from Eptesicus. Thus, our results demonstrated that this species needs to be included in other molecular works for a better understanding of the phylogenetic relationships within the Vespertilioninae subfamily.

Figure 1
Dendrograms of Brazilian species of Chiroptera generated using: (A) Maximum Likelihood (ML) and Bayesian Method (BM) – above nodes/lines = ML bootstrap supports, below nodes/lines = BM supports, above scale = ML distance, below scale = BM distance; (B) Maximum Parsimony (MP) – above nodes/lines = MP bootstrap supports. Molossus molossus was used as outgroup.

Other important results were the monophyly of the Vespertilioninae subfamily (congregates species of Lasiurus, Histiotus and Eptesicus genera) and of the Myotis genus (Myotinae subfamily), with high statistical support. These results were demonstrated in other cytogenetic and molecular studies (Ruedi et al., 2013, as an example). However, regarding the Myotis genus, M. albescens appears as closely related to M. levis and M. nigricans in some studies (Lack et al., 2010, as an example), a fact that was not observed here. Nonetheless, the close phylogenetic relationship among these three species is always observed in analyzes involving Neotropical species (Jones et al., 2002; Ruedi et al., 2013).

Acknowledgements

This work was supported by CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Master Scholarship for G. R. Salomón), FINEP and UNICENTRO.

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Publication Dates

• Publication in this collection
  17 Jan 2022
• Date of issue
  2024

History

• Received
  12 Nov 2021
• Accepted
  02 Jan 2022