Eastern Colombian Páramo Liodessus Guignot, 1939 diving beetles are genetically structured, but show signs of hybridization, with description of new species and subspecies (Coleoptera, Dytiscidae)

Michael Balke; Katja Neven; Adrián Villastrigo; Rodulfo Ospina-Torres; Carlos Prieto; Nicolas Gutierrez Rubiano; Ingrid Lotta; Luisa F. Dueñas; Lars Hendrich

doi:10.3897/zookeys.1143.97461

Abstract

We studied Liodessus diving beetles from six eastern Colombian Páramo areas, as well as from the Altiplano. We discovered a highly characteristic new species, based on male genital morphology, Liodessus santarosita sp. nov., in the Páramo de Guantiva-Rusia. Specimens from the Altiplano around Bogotá, and the Páramos of Almorzadero, Chingaza, Matarredonda, Rabanal y Rio Bogotá and Sumapaz form one clade of genetically similar populations based on mitochondrial Cox1 sequence data. The individuals of this clade are sub-structured according to their geographic distribution. The populations differ from each other mainly in terms of body size and coloration and, at most, subtly in their genital morphology. In two cases, we find putative hybrid populations between Altiplano and Páramo areas. We suggest that the different Páramo populations are in an early phase of speciation, and perhaps already genetically isolated in some cases. They are here assigned subspecies status to highlight these ongoing processes pending more comprehensive geographic sampling and use of genomic data. We refer to this clade as the Liodessus bogotensis complex, containing Liodessus b. bogotensis Guignot, 1953; Liodessus b. almorzadero ssp. nov.; Liodessus b. chingaza ssp. nov.; Liodessus b. lacunaviridis Balke et al., 2021, stat. nov.; Liodessus b. matarredonda ssp. nov., and Liodessus b. sumapaz ssp. nov.

Keywords

Colombia, Dytiscidae, eastern cordillera, Liodessus, new species, new subspecies, Páramo

Introduction

Diving beetles of the genus Liodessus Guignot, 1939 (Guignot 1939) belong to the tribe Bidessini and occur in the New World as well as the Afrotropical Region (Biström 1988; Nilsson and Hájek 2022). They are typically smaller than 3 mm and inhabit a variety of mainly lotic habitats. Andean species reach altitudes of nearly 5,000 m, where they are the most abundant aquatic beetles (Balke et al. 2020a, b).

However, diving beetles from the high altitudes of the Puna and Páramo regions have remained poorly studied until recently. Since 2019, as the result of research and training cooperation between our institutions, we regularly provide updates on the high-altitude fauna (e.g. Megna et al. 2019; Balke et al. 2020a, b). It has become apparent that many more new species of Liodessus remain to be discovered in the vast Andean highland ecosystems; most of these undiscovered species are likely endemic to one or a few Páramo or Puna areas. To address this integrating morphological as well as molecular evidence, we are building a DNA sequence-based platform for the study of these insects, using the Barcode of Life Data System (BOLD) of the Canadian Centre for DNA Barcoding and the 5' mitochondrial Cox1 gene fragment (www.boldsystems.org; Ratnasingham and Hebert 2007). The rationale behind this approach was in detail described by Balke et al. (2021b). The public COLLI project on BOLD currently contains 276 sequences for the 5' end of the cytochrome c oxidase subunit I for 14 species.

Here, we focus on taxa from the eastern mountain range of Colombia, specifically the Altiplano around Bogotá, and the Páramos of Almorzadero, Chingaza, Guantiva-Rusia, Matarredonda, Rabanal y Rio Bogotá, and Sumapaz.

To date, three Liodessus species have been reported from the eastern Colombian branch of the Andes; i.e., L. bogotensis, L. lacunaviridis, and L. picinus (Megna et al. 2019; Balke et al. 2021a, b).

The goals of this publication are (1) to present a new species with highly characteristic male genitalia from the Páramo de Guantiva-Rusia and (2) to reveal a complex of apparently subspecific taxa within Liodessus bogotensis Guignot, 1953. The treatment of this complex is based on mitochondrial Cox1 sequence data as well as male genitalia, where specimens are genetically well structured, with various degrees of supporting morphological features such as genital shape, and body size, shape, and coloration. We here treat them as a complex of subspecies to flag the studied populations, until further genomic evidence and increased geographic sampling help in determining whether these should be considered separate species. Whatever the status of these taxa, this study shows that the Páramo areas studied harbor unique biodiversity worthy of adequate conservation efforts.

Materials and methods

Study area and map generation

Our sampling includes specimens collected in the Altiplano around Bogotá and the Páramos of Almorzadero, Chingaza, Matarredonda, Rabanal y Río Bogotá, and Sumapaz (Fig. 1). The naming of Páramo follows the “Atlas de páramos de Colombia” (Morales et al. 2007). Our map was generated with QGIS v. 3.16.4 (http://www.qgis.org) using the Natural Earth raster map as a base map and the cartography of the Colombian Páramos (IAVH 2013).

Figure 1.

Map of sampling localities A overview of northwestern South America showing are of sampling depicted in “B” B colored areas represent districts in Páramo: Santanderes (blue), Boyacá (green), and Cundinamarca (brown).

Acronyms

UNAL Insect collection, Universidad Nacional de Colombia, Bogotá, Colombia;

ZSM SNSB-Zoologische Staatssammlung, München, Germany, temporarily stored for further morphological work.

Codes in the format COL_MB_2022_004 in the studied material sections are ZSM locality codes, and refer to the country of origin (COL = Colombia), collector who organized the fieldwork (MB = Michael Balke), year of collection (2022) and a locality number for the respective collecting event (004).

Morphological descriptions and photography

The description of morphological characters follows our previous work on Liodessus beetles (Balke et al. 2020b).

Images were taken with a Canon EOS R camera. We used Mitutoyo 10× and 20× ELWD Plan Apo objectives for photographing habitus and genital structures, respectively. These were attached to a Carl Zeiss Jena Sonnar 3.5/135 MC. Illumination was with three LED segments SN-1 from Stonemaster (https://www.stonemaster-onlineshop.de). Image stacks were generated using the Stackmaster macro rail (Stonemaster), and images were assembled with Helicon Focus v. 7.61 on a MacPro 2019 with a Radeon Pro 6800X MPX GPU.

DNA analysis

The DNA sequencing and data analysis laboratory protocol follows standard Canadian Centre for DNA Barcoding (CCDB) barcoding procedures (https://ccdb.ca). We delivered tissue samples to CCDB, which were processed and the barcode data (COI-5) uploaded to BOLD systems. We used a simple approach to calculate a neighbor-joining tree (p-distances) in Geneious v. 11.0.4. to determine if newly added entries could be assigned to existing species groups. This approach has been proven helpful and in guiding the morphological descriptive process.

Diagnostic characters

We aligned the 276 COLLI sequences using the “BOLD > sequence analysis > diagnostic characters” option to detect diagnostic characters mentioned below (Table 1). Data were aligned using the MUSCLE algorithm (Edgar 2004), which resulted in a 658-bp alignment that was inspected using Geneious software.

Table 1.

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XLSX

Diagnostic characters in the Cox1 alignment of the COLLI project on BOLD webportal. Above two taxa relative to all other entries in COLLI, and below, within the L. bogotensis complex. Numbers above indicate nucleotide position in the 658 bp alignment.

Taxon, Number of sequences	19	22	25	49	58	59	82	88	91	106	127	206	217	223	232	256	286	290	304	316	325
Liodessus santarosita (6)	C		T	C	T				A			C		T	A	C	T	A
Liodessus bogotensis (55)
L. bogotensis almorzadero (4)			G					A
L. bogotensis bogotensis (21)						T
L. bogotensis chingaza (16)		A								T	C
L. bogotensis lacunaviridis (4)							C												A	A	G
L. bogotensis matarredonda (5)													G
L. bogotensis sumapaz (5)
	331	334	337	352	355	358	412	421	445	472	473	490	511	542	547	548	553	586	616	643	655
Liodessus santarosita		G	T	A		C			C								G	A		T
Liodessus bogotensis					T										C
L. bogotensis almorzadero														T
L. bogotensis bogotensis
L. bogotensis chingaza										G		C									G
L. bogotensis lacunaviridis	G						G	C								T			T
L. bogotensis matarredonda
L. bogotensis sumapaz											A		G

Haplotype network, fixation index, and genetic distance calculation

DNA sequences from the L. bogotensis complex were aligned and haplotypes were inferred using PHASE (Stephens et al. 2001) for 1,000 iterations, considering a thinning parameter of 5 and a burn-in fraction of 10% iterations. A haplotype network (Fig. 2) was reconstructed with TCS software (Clement et al. 2000) using a broad-connection threshold to attain a fully connected network. Visualization of the haplotype network was conducted with tcsBu (Múrias dos Santos et al. 2016) using collecting localities as groups for enhanced visualization.

Figure 2.

Haplotype network for the Liodessus bogotensis complex using the partial gene COI-5.

We also computed the fixation index (F_ST) as a measure of population differentiation. F_ST values ranges from 0 to 1 and are an indication whether populations freely interbreed (panmixis, value of 0) or if gene flow is absent and populations are genetically isolated (value of 1). For this, we used DNAsp v. 6 (Rozas et al. 2017), defining the clusters found in the haplotype network as sets of sequences (Fig. 3). Moreover, minimum genetic divergence between populations were calculated using Geneious v. 11.04.

Figure 3.

Heatmap of pairwise comparison of F_ST values (A) and Minimum genetic divergence (B) among the clusters as defined by the haplotype network.

Results and discussion

A new Liodessus species from Páramo de Guantiva-Rusia

Liodessus santarosita sp. nov.

https://zoobank.org/B86A86A2-282B-4475-B833-3543606E52DC

Fig. 4A–E

Type locality

Santa Rosita, Páramo de Guantiva-Rusia, Boyaca, Colombia.

Holotype : Colombia • ♂; Boyaca, Santa Rosita, El Parador de Gallina; 3,200 m alt.; 7.v.2022; 6.1563, -72.7681; Gutierres, Ospina, & Balke leg.; COL_MB_2022_003; UNAL. Paratypes: Colombia • 165 specimens; same data as holotype; UNAL, ZSM. ZSM specimen imaging number for holotype: ZSM-COL-00127.

Description of holotype

Habitus with slight discontinuity between pronotum and elytra (Fig. 4A); pronotum widest before base (Fig. 4A). Total length 2.1 mm; length without head 1.8 mm; maximum width 1.0 mm.

Figure 4.

Liodessus santarosita sp. nov. A habitus B median lobe, lateral view C median lobe, ventral view D, E parameres lateral view. Scale bar: 2 mm.

Color. Very dark brown to blackish dorsally and ventrally; lighter on lateral pronotum, and bases of meso- and metatibia (Fig. 4A).

Surface sculpture. Head with a few setiferous punctures in front of a distinct occipital line; distinct microreticulation present except on middle of head between eyes (Fig. 4A); posterior to occipital line with distinct microreticulation and a few punctures. Pronotum and elytron shiny, with moderately dense and coarse setiferous punctation (Fig. 4A).

Structures. Head with distinct occipital line, with rounded clypeus. Antenna stout. Pronotum with distinct lateral bead and distinct, long, deep basal striae (Fig. 4A). Elytron with short basal striae.

Genitalia. Median lobe of aedeagus with bulbous main body in lateral view, strongly narrowing at tip and with delicate downwards bent hook; gradually narrowing towards a very narrow, needle-like tip in ventral view (Fig. 4B, C); parameres simple of the “Bidessini” type, 2-segmented (Fig. 4D, E).

Variation

Total length 2.1–2.3 mm (N = 20); length without head 1.8–2.1 mm; maximum width 1.0–1.1 mm. In a few specimens, the elytral plicae are fairly obsolete.

Metathoracic wings short, 2/3 of elytral length, venation visible (in one dissected male paratype).

Female. External morphology as in male.

Etymology

After the village of Santa Rosita, near the type locality. The word “santarosita” is a noun in the nominative singular standing in apposition.

Identification notes

This species differs from all other Liodessus by the needle-like apical part of the median lobe of the aedeagus. In the COLLI sequence database, the species has 19 diagnostic characters different from the other Andean species of the genus (Table 1).

Distribution

Only known from the type locality in the Páramo de Guantiva-Rusia (Fig. 1).

Habitat

Exposed, densely vegetated peatland swamp.

The Liodessus bogotensis complex

Identification notes. Beetles of this complex are characterized by the following combination of characters: Liodessus from the eastern cordillera of Colombia; occipital line usually present, although faint in some specimens; coloration overall dark to bright due to more extensive yellowish or orange dorsal markings (Figs 5–7); median lobe of aedeagus forming a simple curve in lateral and ventral views (Figs 8–10).

Figure 5.

Variation in size and dorsal coloration of subspecies of Liodessus bogotensis A Liodessus b. bogotensis B Liodessus b. almorzadero C Liodessus b. chingaza. Scale bar: 2 mm.

Figure 6.

Variation in size and dorsal coloration of subspecies and a hybrid of Liodessus bogotensis A Liodessus b. matarredonda B hybrid specimens of L. b. bogotensis and L. b. matarredonda. Scale bar: 2 mm.

Figure 7.

Variation in size and dorsal coloration of subspecies of Liodessus bogotensis A Liodessus b. lacunaviridis B Liodessus b. sumapaz. Scale bar: 2 mm.

Figure 8.

Median lobes in lateral view of Liodessus species A L. bogotensis bogotensis B L. b. almorzadero C L. b. chingaza. Scale bar: 0.2 mm.

Figure 9.

Median lobes in lateral view of Liodessus subspecies A L. bogotensis lacunaviridis B L. b. matarredonda C L. b. sumapaz. Scale bar: 0.2 mm.

Figure 10.

Tips of median lobes in ventral view of Liodessus subspecies A L. bogotensis bogotensis B L. bogotensis almorzadero C L. bogotensis chingaza D L. b. lacunaviridis E L. b. matarredonda F L. b. sumapaz. Scale bar: 0.1 mm.

In the COLLI sequence database, L. bogotensis has two diagnostic characters different from the other Andean species of the genus (Table 1).

Phylogeographic evidence. We detected a marked phylogeographic structure among subpopulations of this complex (Figs 2, 3). Six haplotype clusters were recovered, representing five areas in the Páramo as well as the Altiplano around Bogotá up to an altitude of 3,100 m.

Some specimens exhibit intermediate morphological characteristics between the subspecies L. b. bogotensis and L. b. matarredonda and may be hybrids. They were clustered together with L. b. bogotensis. The low F_ST value (F_ST = 0.095) suggests gene flow between the L. b. bogotensis and L. b. matarredonda populations.

The fixation index, on the other hand, revealed relatively higher population differentiation (F_ST > 0.7) among all other clusters except Liodessus b. chingaza (F_ST < 0.7), which might suggest enhanced connectivity of Liodessus b. chingaza with other subspecies. However, the genetic divergence of Liodessus b. chingaza from the other subspecies is moderate to high compared to the other clusters (more than 2.4% in all cases), which may indicate an ongoing speciation process.

Minimum genetic distances between populations (Fig. 3B) show no divergence between the putative L. b. bogotensis × matarredonda hybrid and L. b. bogotensis. Most other subpopulations diverge from each other by 1.2–4.0%, with L. b. lacunaviridis the most divergent subpopulation (2.1–4.0%), which is in accordance with its greater genetic isolation (Fig. 1). Cox1 divergence above 2% is widely assumed to be a rough indicator for the presence of distinct species, from a simple DNA-sequencing perspective (Hebert et al. 2003). However, lineage idiosyncrasies might always blur this picture (Hendrich et al. 2010).

Here, we suggest that ongoing speciation or very young species might be present in Liodessus from the eastern cordillera of Colombia. Making this distinction clearly requires greater sampling over the geographic area and the addition of nuclear markers. This study again underpins the high potential and possible pitfalls of using genetic data for taxonomy on the one hand, and the need for morphological examination on the other hand (Meier et al. 2022).