Phylogenetic affinities of the non-cyclostome subfamilies Amicrocentrinae and Dirrhopinae (Hymenoptera, Braconidae) confirmed by ultraconserved element data

The subfamilies Amicrocentrinae and Dirrhopinae (Hymenoptera, Braconidae) are two small, monogeneric braconid subfamilies whose species exclusively attack lepidopteran larvae. The phylogenetic placement of Amicrocentrinae as a member of the helconoid complex of subfamilies has been supported by morphological and nuclear Sanger sequence data. The subfamilial status of Dirrhopinae on the other hand has been subject to debate, although it has been suggested as closely related to the microgastroid complex based on morphology only. Here we generated for the first time genomic ultraconserved element data for members of the above subfamilies (Amicrocentrum seyrigi van Achterberg and Dirrhope americana Muesebeck) to assess their phylogenetic affinities using exhaustive taxon sampling that includes all but one of the currently valid braconid subfamilies. Our results strongly confirm the placement of both taxa within the non-cyclostome helconoid and microgastroid complexes, respectively

Four new species of Triraphis Ruthe, 1855 (Braconidae, Rogadinae) from a Mexican tropical dry forest and morphological descriptions of T. bradzlotnicki Sharkey, 2021 and T. davidwahli Sharkey, 2021

The koinobiont endoparasitoid genus Triraphis Ruthe, 1855 (Rogadinae Foerster, 1863) is a group of braconid wasps that contains 74 species distributed along the Nearctic, Neotropical, Oriental and Palearctic regions. We amplified a fragment of the cytochrome oxidase subunit I (COI) for 19 specimens of Triraphis from the Chamela Biological Station (CBS), a region mainly composed of tropical dry forest near the Pacific coast of Jalisco, Mexico. Based on genetic distances among specimens of Triraphis from the CBS and all COI sequences of BINs assigned to Triraphis and Rogas Nees, 1819 available in the BOLDSYSTEMS database, we identified three clusters in the CBS that correspond with T. bradzlotnicki Sharkey, 2021, T. davidwahli Sharkey, 2021 and T. defectus Valerio, 2015, which were previously described from Costa Rica. Based on morphology, we identified individuals of T. fusciceps Cresson, 1869 and provided COI sequences of this species for the first time. Four genetic clusters of Triraphis correspond to four new species that are described here: T. kardia sp. nov., T. ocellatus sp. nov., T. divergens sp. nov. and T. luzabrilae sp. nov. Since T. bradzlotnicki and T. davidwahli were exclusively described with molecular data (COI), we morphologically described them based on Mexican specimens

Molecular evidence of hybridization in sympatric populations of the <i>Enantia jethys</i> complex (Lepidoptera: Pieridae)

<div><p>Hybridization events are frequently demonstrated in natural butterfly populations. One interesting butterfly complex species is the <i>Enantia jethys</i> complex that has been studied for over a century; many debates exist regarding the species composition of this complex. Currently, three species that live sympatrically in the Gulf slope of Mexico (<i>Enantia jethys</i>, <i>E</i>. <i>mazai</i>, and <i>E</i>. <i>albania</i>) are recognized in this complex (based on morphological and molecular studies). Where these species live in sympatry, some cases of interspecific mating have been observed, suggesting hybridization events. Considering this, we employed a multilocus approach (analyses of mitochondrial and nuclear sequences: <i>COI</i>, <i>RpS5</i>, and <i>Wg;</i> and nuclear dominant markers: inter-simple sequence repeat (ISSRs) to study hybridization in sympatric populations from Veracruz, Mexico. Genetic diversity parameters were determined for all molecular markers, and species identification was assessed by different methods such as analyses of molecular variance (AMOVA), clustering, principal coordinate analysis (PC_oA), gene flow, and <i>PhiPT</i> parameters. ISSR molecular markers were used for a more profound study of hybridization process. Although species of the <i>Enantia jethys</i> complex have a low dispersal capacity, we observed high genetic diversity, probably reflecting a high density of individuals locally. ISSR markers provided evidence of a contemporary hybridization process, detecting a high number of hybrids (from 17% to 53%) with significant differences in genetic diversity. Furthermore, a directional pattern of hybridization was observed from <i>E</i>. <i>albania</i> to other species. Phylogenetic study through DNA sequencing confirmed the existence of three clades corresponding to the three species previously recognized by morphological and molecular studies. This study underlines the importance of assessing hybridization in evolutionary studies, by tracing the lineage separation process that leads to the origin of new species. Our research demonstrates that hybridization processes have a high occurrence in natural populations.</p></div

Consensus trees obtained by Bayesian inference for the <i>Enantia jethys</i> complex based on DNA sequences.

<p>(A) <i>Wg</i>, (B) <i>RpS5</i>, and (C) <i>COI</i>. Branch colors of the phylogenetic trees correspond to morphospecies identification: <i>E</i>. <i>albania</i> (red), <i>E</i>. <i>mazai</i> (blue), and <i>E</i>. <i>jethys</i> (green). Asterisks indicate the genetic profile obtained by Structure analysis based on ISSR molecular markers, where a red asterisk indicates a genetic component characteristic of <i>E</i>. <i>albania</i>, a blue asterisk indicates a genetic component of <i>E</i>. <i>mazai</i>, and a green asterisk indicates a genetic component of <i>E</i>. <i>jethys</i>. Presence of two different asterisks indicates an individual with mixed genetic components, based on Structure results.</p

Identification of admixture and no-admixture (i.e., hybrids and non-hybrids) individuals of the <i>Enantia jethys</i> complex through ISSR profiles using (AG)₈Y primers.

<p><i>Enantia albania</i> (<i>E</i>. <i>a</i>), <i>Enantia jethys</i> (<i>E</i>. <i>j</i>), <i>Enantia mazai</i> (<i>E</i>. <i>m</i>). Hybrid individuals identified by Structure analysis: hybrid between <i>E</i>. <i>albania</i> and <i>E</i>. <i>jethys</i> (<i>E</i>. <i>a</i> × <i>E</i>. <i>j</i>), hybrid between <i>E</i>. <i>albania</i> and <i>E</i>. <i>mazai</i> (<i>E</i>. <i>a</i> × <i>E</i>. <i>m</i>), and hybrid among the three morphospecies (<i>E</i>. <i>a</i> × <i>E</i>. <i>j</i> × <i>E</i>. <i>m</i>). Information about morphospecies (line 1) and information about genetic profile (line 2) obtained by Structure analysis.</p

Characteristics of ISSR primers used for studying the <i>Enantia jethys</i> complex.

<p>Characteristics of ISSR primers used for studying the <i>Enantia jethys</i> complex.</p

Indices of haplotype diversity for the three morphospecies of the <i>Enantia jethys</i> complex based on mitochondrial (<i>COI</i>) and nuclear (<i>RpS5</i> and <i>Wg</i>) DNA sequences.

<p>Indices of haplotype diversity for the three morphospecies of the <i>Enantia jethys</i> complex based on mitochondrial (<i>COI</i>) and nuclear (<i>RpS5</i> and <i>Wg</i>) DNA sequences.</p

Tree obtained by distance analysis for the <i>Enantia jethys</i> complex using ISSR data.

<p>(A) Parsimony tree for the three morphospecies: <i>E</i>. <i>albania</i> (red); <i>E</i>. <i>mazai</i> (blue); and <i>E</i>. <i>jethys</i> (green). Colored asterisks correspond to the genetic profile obtained by Structure analysis. To improve the understanding of the tree, we present a zoom of each section of the tree corresponding to each morphospecies: (A1) <i>E</i>. <i>albania</i> zone of the tree (red section); (A2) <i>E</i>. <i>mazai</i> (blue section); and (A3) <i>E</i>. <i>jethys</i> (green section).</p

Female and male phenotypes for each morphospecies of the <i>Enantia jethys</i> complex in Mexico.

<p><i>Enantia albania</i> (A), <i>Enantia jethys</i> (B), <i>Enantia mazai</i> (C), males (1), females (2) (photos by JM Jasso-Martínez).</p