Leeches from Chiapas, Mexico, with a new species of Erpobdella (Hirudinida, Erpobdellidae)

AMERICAN MUSEUM NOVITATES

Number 3895, 15 pp.

March 12, 2018

Leeches from Chiapas, Mexico, with a new species of
Erpobdella (Hirudinida: Erpobdellidae)

MICHAEL TESSLER , 1 ’ 2 ' 3 MARK E. SIDDALL , 1 ' 2
AND ALEJANDRO OCEGUERA-FIGUEROA 4

ABSTRACT

Freshwater leeches were collected from Chiapas, Mexico. Among these collections and
prior records, a total of nine species were found, comprising six genera and four families. One
species, Diestecostoma octannulata, represents a new record for Mexico and two species, Helob-
della elongata and H. octatestisaca, represent new records for Chiapas. Additionally, a new
species of Erpobdella was discovered from a single stream in the park El Arcotete near San
Cristobal de las Casas. Here it is described as Erpobdella adani and it is morphologically dis¬
tinguished from Mexican Erpobdella species by the combination of having the whole body
strongly dorsoventrally flattened, three annuli between gonopores, an enlarged male gonopore,
and no preatrial loops for the male reproductive system. Based on sequence data (mitochon¬
drial cytochrome c oxidase subunit I and 12S; nuclear 18S), this new species appears most
closely related to a clade formed by E. coastalis and E. ochoterenai. Both mitochondrial cyto¬
chrome c oxidase subunit I molecular barcodes and morphological descriptions of the collected
species are presented.

1 Division of Invertebrate Zoology, American Museum of Natural History.

2 Sackler Institute for Comparative Genomics, American Museum of Natural History.

3 Richard Gilder Graduate School, American Museum of Natural History.

4 Laboratorio de Helmintologia, Instituto de Biologia, Universidad Nacional Autonoma de Mexico. Ciudad
de Mexico, Mexico.

Copyright © American Museum of Natural History 2018

ISSN 0003-0082

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INTRODUCTION

Mexico is well known as one of the worlds biodiversity hotspots (Ramamoorthy, 1993).
Although collecting efforts for leeches have grown slowly, it is beginning to become clear that
leeches are also diverse in Mexico. In the past decade a massive effort to collect leeches through¬
out the central states of Mexico has taken place (Oceguera-Figueroa and Leon-Regagnon, 2014).
These explorations have been fruitful and clearly establish Mexico as ripe for leech discovery, with
many endemics uncovered. Furthermore, these leeches are phylogenetically diverse, with repre¬
sentatives from over 70% of leech families (Oceguera-Figueroa and Leon-Regagnon, 2014).

No other area in North America has had so many recent leech species discoveries, and
there is no sign of this rapid rate of discovery slowing (Oceguera-Figueroa and Leon-Rega¬
gnon, 2014). Concerted efforts to further collect and systematize Mexican leech diversity
have filled in many of the gaps in records, collections, and undescribed diversity, especially
from Central Mexico. Chiapas, the southernmost state of Mexico, has had few surveying
efforts. Still, this work has resulted in interesting discoveries of endemic species, such as the
mammalophilic leech Pintobdella chiapasensis Caballero, 1957, known only from Custe-
peques and Lagunas de Montebello (Caballero, 1957; Phillips et al., 2010), and a turtle¬
feeding species, Placobdella ringueleti Lopez-Jimenez and Oceguera-Figueroa, 2009, from
Laguna Belgica, Bonampak, Rancho Alexandria, Malpaso Dam, Tuxtla Gutierrez, and
Reforma. Furthermore, Haementeria acuecueyetzin Oceguera-Figueroa, 2008, was found
parasitizing a manatee in Palenque, Chiapas, which is the first and only record of a leech
feeding on a manatee (Perez-Flores et al., 2016). In addition to these records, two species of
leeches are known from the Coleccion Nacional de Helmintos (CNHE), Instituto de Biologfa,
UNAM: Erpobdella triannulata Moore, 1908, from Lagunas de Montebello and Helobdella
socimulcensis Caballero, 1931, from Presa Belisario Dominguez.

Overall, Chiapas has largely remained unexplored (Oceguera-Figueroa and Leon-Rega¬
gnon, 2014). In the present study, we provide a brief description of the morphology and genetic
barcodes—mitochondrial cytochrome c oxidase subunit I fragment (coxl)—for each species
newly collected in Chiapas. We present new localities for three species, a new country record,
as well as the description and phylogenetic placement of a new species of macrophagous leech
of the genus Erpobdella.

MATERIALS AND METHODS

Leeches were collected in 2014 and 2015 from localities across Chiapas (fig. 1) under the
permit SEMARNAT 12099 to A.O.-F. Collection techniques primarily followed two standard
survey strategies: (1) bare-legged wading in water to attract blood-feeding leeches; and (2) a
more active search for both blood-feeding and non-blood-feeding leeches by flipping over
rocks, sticks, and vegetation both in and at the edge of the water. Both lentic and lotic sites
were checked, but a greater focus was made on lentic sites due to their generally higher leech
diversity. Additionally, terrestrial leeches in the Xerobdellidae were sought in moist forests by
flipping over rocks and logs. Specimens were gradually relaxed in water with increasing con¬
centrations of ethanol, and eventually fixed in 70%-90% ethanol. Collected leeches were identi-

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Campeche

1. Diestecostoma octannulata

2. Erpobdella adani, n. sp.

3. Erpobdella triannulata

4. Haementeria acuecueyetzin

5. Helobdella elongata

6. Helobdella octatestisaca
1. Helobdella socimulcensis

8. Pintobdella chiapasensis

9. Placobdella ringueleti

100 km

FIGURE 1. Map of the State of Chiapas, Mexico, with the collecting localities of leeches.

fied and photographed under a stereomicroscope. In addition, in this study we include samples
collected by other biologists as well as previously published records.

At least one specimen from each species collected was genetically barcoded with coxl. DNA
extractions, amplifications, and sequencing protocols followed prior studies (Apakupakul et al.,
1999; Borda and Siddall, 2004). For the new species of Erpobdella we amplified a longer fragment
of coxl using HHCOl (developed at the AMNH) 5'-GCTGCAAAAATRGCAAATACTGC-3'
instead of HC02198, but otherwise kept identical protocols (Apakupakul et al., 1999). For the
new species, we also amplified the mitochondrial 12S following Simon et al. (1994) using 12Sa
and 12Sb primers and nuclear 18S (amplified and sequenced using primers “A,” “B,” and “Y”)
with the protocols found in Borda and Siddall (2004) and Apakupakul et al. (1999), respectively.
Sequences were reconciled in Geneious version 6.1.8 (Biomatters Limited).

To determine the phylogenetic position of the new species within Erpobdella, a matrix was
constructed for those Erpobdella species with sequence data available for coxl, 12S, and/or 18S
(table 1). Sequences were aligned using MUSCLE (Edgar, 2004a, 2004b) through the “muscle”
package in R (www.r-project.org/). Both parsimony via TNT (GolobofF et al., 2008) and maxi¬
mum likelihood (ML) via Garli (Zwickl, 2006) were then used to infer the phylogeny. For ML
reconstruction, loci were partitioned using models found with jModelTest 2.1.4 (Darriba et al.,
2012). For both inferences, support was assessed using 1000 bootstrap pseudoreplicates, sum¬
marized on the ML tree using SumTrees (Sukumaran and Holder, 2010). Trees were visualized
in FigTree vl.4.2 (Rambaut, 2014). Additionally, coxl sequences were used to calculate genetic
distances with the Kimura two-parameter using PAUP* 4.0b 10 (Swofford, 2002).

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TABLE 1. Molecular data used to determine the phylogenetic placement of Erpobdella adani within the
genus Erpobdella. GenBank accession numbers are given for each sequence.

Family

Species

Coxl

12S

18S

Country

Erpobdellidae

Erpobdella adani n. sp. 1

MG745144

MG745141

MG745138

Mexico

Erpobdella adani n. sp. 2

MG745145

MG745142

MG745139

Mexico

Erpobdella adani n. sp. 3

MG745146

MG745143

MG745140

Mexico

Erpobdella annulata

HQ336345

—

HQ336379

USA

Erpobdella borisi

KP749904

—

—

Iran

Erpobdella bucera

AF116024

AF462026

AF115998

USA

Erpobdella bychowskii

DQ009667

AF169372

—

Slovenia

Erpobdella coastalis 1

AY425460

AY425442

AY425478

USA

Erpobdella coastalis 2

—

HQ336354

HQ336381

USA

Erpobdella dubia

AF116023

AF462022

AF 115997

USA

Erpobdella haskonis

DQ009668

—

—

Germany

Erpobdella [Trocheta] intermedia

DQ009669

—

Germany

Erpobdella japonica 1

AB679654

AB679655

A P,6636-IS

Japan

Erpobdella japonica 2

AF116026

AF462023

AF116000

Korea

Erpobdella johanssoni

HM246605

—

—

Spain

Erpobdella krilata

HM246629

—

—

Albania

Erpobdella latestriata

HM246610

—

—

Greece

Erpobdella [Dina] lepinja

HM246597

—

—

Macedonia

Erpobdella lineata 1

—

AF099952

AF099950

Denmark

Erpobdella lineata 2

HM246611

—

—

Macedonia

Erpobdella [Dina] lyhnida

HM246595

—

—

Macedonia

Erpobdella melanostoma

AF116025

AF462027

AF115999

USA

Erpobdella mexicana 1

DQ235595

DQ235585

DQ235605

Mexico

Erpobdella mexicana 2

DQ235601

DQ235591

DQ235611

Mexico

Erpobdella mexicana 3

DQ235597

DQ235587

DQ235607

Mexico

Erpobdella monostriata

DQ009665

—

—

Germany

Erpobdella montezuma

GQ368760

GQ368820

GQ368802

USA

Erpobdella nigricolis

DQ009664

-

Germany

Erpobdella obscura

AF003273

AF462028

All 16003

Canada

Erpobdella ochoterenai 1

DQ235603

DQ235593

DQ235613

Mexico

Erpobdella ochoterenai 2

DQ235599

DQ235589

DQ235609

Mexico

Erpobdella ochoterenai 3

DQ235600

DQ235590

DQ235610

Mexico

Erpobdella octoculata 1

AF003274

AF099954

AF116001

France

Erpobdella octoculata 2

HQ336344

—

HQ336378

Uzbekistan

Erpobdella [Dina] ohridana

HM246633

—

—

Macedonia

Erpobdella [Dina] cf. profunda

HM246581

—

—

Macedonia

Erpobdella [Trocheta] pseudodina

EF125041

—

—

Germany

Erpobdella punctata 1

HQ336346

HQ336352

HQ336380

Canada

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Family

Species

Coxl

12S

18S

Country

Erpobdella punctata 2

AF003275

AF462024

AF116002

Canada

Erpobdella subviridis

—

AF169374

—

Croatia

Erpobdella svilesta

HM246598

—

—

Macedonia

Erpobdella testacea

AF116027

AF462025

AF 116003

France

Erpobdella triannulata 1

DQ235602

DQ235592

DQ235612

Mexico

Erpobdella triannulata 2

DQ235604

DQ235594

DQ235614

Mexico

Erpobdella triannulata 3

HQ336347

HQ336353

—

Mexico

Erpobdella vilnensis

DQ009663

—

—

Germany

Gastrostomobdellidae

Gastrostomobdella monticola

AB679656

AB679657

AB663649

Malaysia

Orobdella dolichopharynx

AB679680

AB679681

AB663665

Japan

Salifidae

Barbronia weberi

DQ235598

DQ235588

DQ235608

Mexico

Linta be

AY786460

—

AY786466

Madagascar

Mimobdella japonica

AB679658

AB679659

AB663650

Japan

Odontobdella blanchardi

AB938004

AB937995

AB663651

Japan

Salifa motokawai

LC029431

LC029432

LC029434

Vietnam

SYSTEMATICS

Order Arhynchobdellida Blanchard, 1894
Family Erpobdellidae Blanchard, 1894

Erpobdella adani, n. sp.

Figures 2-6

Material Examined: Fifteen specimens collected in Rio Fogotico, in Arcotete, Chiapas,
Mexico, on January 23, 2016, by Michael Tessler, Samantha Contreras, Jairo Arroyave, and
Alejandro Oceguera-Figueroa. Specimens were found beneath submerged rocks in moderately
flowing water. Non-blood-feeding (macrophagous).

Description: External morphology based on 15 specimens. Body strongly dorsoven-
trally flattened, vermiform, slender. Dorsal surface dark gray, ventral surface lighter; some
specimens with three longitudinal lines of black pigment, others lacking lines. Dorsal surface
smooth, without conspicuous papillae or other sensory structures; ventral surface of some
specimens covered with minute papillae (figs. 2, 3). Average body length 51 mm (35-64
mm), average body width 4 mm (2-5 mm). Complete somite five annulate. Clitellum con¬
spicuous, thick both dorsally and ventrally, comprising 15 annuli, from X b5 to XIII a2. Male
gonopore at XII b2/a2, female gonopore XII/XIII; three annuli between gonopores (fig. 4).
Male gonopore large, conspicuous, and surrounded by an integumental disc; female gono¬
pore small. Three pairs of eyespots in the classic “erpobdellid” arrangement with the anterior
labial pair on III and two buccal pairs at IV. Mouth wide, occupying entire anterior sucker;
anterior lip blunt (fig. 5). Anus large, on XXVI/XXVII. Posterior sucker weakly developed,

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FIGURES 2-6. Erpobdella adani. 2. Holotype, dorsal view. 3. Holotype, ventral view. 4. Paratype, male gono-
pore (black arrow), female gonopore (white arrow). 5. Paratype, ventral anterior end, anterior sucker. 6.
Illustration of the male (dark gray) and female (light gray) reproductive systems, atrial horns (ah), ejaculatory
duct (ed), ventral nerve cord (nc), ovary (ov); testisacs not shown.

ventrally directed, narrower than the posterior part of the body. Somites I—III unianulate,
IV-V biannulate, VI three annulate, VII four annulate, VIII-XXIV five annulate, XXV three
annulate, XXVI biannulate, XXVII unianulate.

Internal morphology based on the dissection of three specimens. Agnathous, pharynx
strepsilaematous, muscular, extending to XIII/XIV. Crop tubular, acaecate, extending from XIV
to XIX/XX; intestine acaecate extending to XXVI/XXVII. Male medial reproductive system
with paired atrial horns directed anterolaterally, without preatrial loops (fig. 6). Ejaculatory
ducts tubular, extending posteriorly to XVIII/XIX, continuing with multiple testisacs (forming
grapelike clusters) reaching XXIV. Female medial reproductive system formed by tubular ova¬
ries with a lateral first portion in XIII and then joining the median line of the body mass
extending to XVI/XVII where the ovaries redirect anteriorly to XIII and then they follow the
distribution of the lateral first portion of the ovaries ending laterally at XII/XIII (fig. 6).

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82 —

— Erpobdella japonica 2

— Erpobdella octoculata 2

l 12

Gastriostomobdella monticola

Orobdella dolichopharynx

Barbronia weberi

■ Salifa motokawai
■ Odontobdella blanchardi

• Mimobdella japonica

Erpobdella intermedia

Erpobdella lineata 2
Erpobdella svilesta
Erpobdella cf. profunda
Erpobdella lyhnida
Erpobdella ohridana
Erpobdella lepinja
Erpobdella krilata
Erpobdella obscura
Erpobdella dubia

Erpobdella melanostoma
Erpobdella punctata I
Erpobdella punctata 2
Erpobdella montezuma
Erpobdella annulata

Erpobdella bucera
97 |— Erpobdella mexicana 2

J ' - Erpobdella mexicana 1

* Erpobdella mexicana 3

" i— Erpobdella triannulata 3

J ' - Erpobdella triannulata 2

• - Erpobdella triannulata 1

Erpobdella adani, n. sp. 1
Erpobdella adani, n. sp. 2
Erpobdella adani, n. sp. 3

■ Erpobdella johanssoni

• Erpobdella latestriata

I Erpobdella ochoterenai 3

- Erpobdella ochoterenai 1

- Erpobdella coastalis 1

Erpobdella coastalis 2

Erpobdella lineata 7

• Erpobdella ochoterenai 2

0.2

FIGURE 7. Maximum likelihood phylogenetic reconstruction of the family Erpobdellidae focused on establishing
the closest relatives to Erpobdella adani. Support values are based on 1000 bootstrap pseudoreplicates.

Etymology: This species is named to remember Adan Enrique Gomez Gonzalez
(1980-2018). Adan was an enthusiastic biologist with a deep passion and knowledge
of biodiversity from Mexico, in particular from Chiapas, where he was born and where
the new species of Erpobdella was collected.

Holotype: Coleccion Nacional de Invertebrados, Instituto de Biologia, UNAM (catalog
number 21).

Paratypes: Eleven specimens, Coleccion Nacional de Invertebrados, Instituto de Biologia,
UNAM (catalog number 22); three specimens, American Museum of Natural History (AMNH_
IZC 250222; GenBank accession numbers presented in table 1).

Type Locality: Rio Fogotico, Arcotete, Chiapas, Mexico (16°44'46.5"N, 92°34'17.24"W;
2298 m).

Phylogenetic Analyses: The three loci reviewed did not differ for the three Erpobdella
adani specimens sequenced. Erpobdella adani was found nested in an exclusively North Ameri¬
can clade. It was found to be sister to an E. coastalis/E. ochoterenai Caballero, 1932, clade with
moderate support (fig. 7). This combined clade was sister to E. mexicana (Duges, 1876) in

AMERICAN MUSEUM NOVITATES

NO. 3895

parsimony and sister to an E. mexicana/E. triannulata clade in ML analyses; however, these
relationships had weak support in both analyses.

More generally, the phylogenetic inference resulted in two principal clades within the
genus Erpobdella; these results are similar to previous analyses (Siddall, 2002; Oceguera-
Figueroa et al., 2005, 2011). For both analyses, the two specimens of E. lineata (O.F. Muller,
1774) are not sister; E. lineata 2 (coxl data only; specimen from Macedonia) grouped with the
principally European clade, while E. lineata 1 (12S and 18S data only; specimen from Den¬
mark) varied in its positioning.

Remarks: Only three other species of Erpobdella are known from Mexico: E. mexicana, E.
ochoterenai, and E. triannulata, with only E. triannulata known from Chiapas. The new species,
E. adani, is distinguished from E. mexicana and E. triannulata based on the male reproductive
structures. While E. mexicana and E. triannulata have ejaculatory ducts forming a preatrial
loop extending to XI before entering the atrial horns, in E. adani the preatrial loop is absent
(fig. 6). In addition, E. mexicana has two annuli separating the gonopores while E. adani has
three. In Central America, including Costa Rica, Guatemala, and Honduras (Oceguera-
Figueroa and Pacheco-Chaves, 2012; Cornejo et al., 2015), only E. triannulata has been reported
from this region, the southern limit of the distribution of the species of the genus.

Erpobdella ochoterenai, from Central Mexico, and E. coastalis and E. microstoma (Moore, 1901),
both from the southeastern United States, are morphologically the most similar species to Erpob¬
della adani. These species have three annuli between gonopores and lack preatrial loops of the
ejaculatory ducts. However, in E. adani the body is strongly dorsoventrally flattened, from the
preclitellar region to the posterior sucker. This characteristic is constant in the specimens studied
and apparently not related to the process of narcotization-fixation. This contrasts with E. ochotere¬
nai, E. coastalis, and E. microstoma, which are terete for the anterior two thirds of the body and
dorsoventrally flattened only for the last third of the body (Moore, 1945; A.O.-F., personal obs.).
Additionally, E. adani has the male gonopore unusually enlarged, in some cases occupying almost
all of b2 and half of a2 of somite XII, a character not seen in other members of the genus.

The average coxl genetic distances for samples of the new species is 13% divergent from
Erpobdella ochoterenai and 18% from E. coastalis, values that independent of morphology indi¬
cate that both groups likely represent separate species.

Other Taxa

Order Arhynchobdellida Blanchard, 1894
Family Erpobdellidae Blanchard, 1894
Erpobdella triannulata Moore, 1908
Figures 8-9

The presence of three pairs of eyespots, two pairs of longitudinal lines on the dorsal surface,
three annuli between gonopores, ejaculatory ducts forming preatrial loops, and medium body

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FIGURES 8-11. Arhynchobdellida from Chiapas, Mexico. Erpobdella triannulata : 8. dorsal view, 9. ventral
view. Diestecostoma octannulata: 10 . dorsal view, 11 . ventral view.

size correspond to the description of Erpobdella triannulata (Oceguera-Figueroa et al., 2005).
Found on the underside of submerged rocks and roots of aquatic plants. Non-blood-feeding
(macrophagous). New locality records: Palenque (17°29'10"N, 92°01'10"W; 77 m), four speci¬
mens collected March, 2015, by J. Perez-Flores, (CNI 23); Osumacinta Dam (16°57'47.5"N,
93°06'21.82"W; 207 m), 22 specimens collected January 24, 2016, by M. Tessler, J. Arroyave, S.
Contreras, and A. Oceguera-Figueroa, (CNHE 24). GenBank coxl barcode sequence acces¬
sions: MG821606 and MG821607.

Family Xerobdellidae Moore, 1946
Borda, Oceguera-Figueroa, and Siddall, 2008
Diestecostoma octannulata Moore, 1946
Figures 10-11

The presence of five eyespot pairs arranged in a parabolic arc (first four pairs on contiguous
annuli and two annuli separating fourth and fifth pair), complete somite eight annulate (16 + 1/2
annuli between gonopores) is consistent with the description of Diestecostoma octannulata Moore,
1946. Found in a rotten tree under the bark. Terrestrial leech; presumably non-blood-feeding (mac¬
rophagous). New country record; new state record; new locality record: Mexico, Volcan Tacana, la
Caracola (15°06'19.39"N, 92°05'47.27"W; 2452 m), one specimen collected July, 25, 2015, by Ale¬
jandro Oceguera-Figueroa (CNI 15). GenBank coxl barcode sequence accession: MG821605.

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FIGURES 12-15. Rhynchobdellida from Chiapas, Mexico. Helobdella elongata : 12 . dorsal view, 13 . ventral
view. Helobdella octatestisaca: 14 . dorsal view, 15 . ventral view.

Order Rhynchobdellida Blanchard, 1894
Family Glossiphoniidae Vaillant, 1890
Helobdella elongata Castle, 1900
Figures 12-13

The absence of a chitinous nuchal scute; presence of an unpigmented, nonpapillated, and sub-
cylindrical body in cross section; and weak anterior and posterior suckers all correspond to the
description of Helobdella elongata. Specimens lacked eyespots, a characteristic noticed before for some
individuals of this species from other localities (Klemm, 1982). Found on the underside of submerged
rocks and accidentally attached to skin. Non-blood-feeding (liquidosomatophagous). Coxl genetic

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distances of H. elongata from Chiapas are around 10% divergent from other samples of the same
species from other localities. This suggests that samples from Chiapas may represent an undescribed
species. New state record; new locality records: Tres Lagunas, Chiapas (16°50'33.9"N, 91°08'46.69"W;
370 m) 4 specimens collected January 27, 2016 by M. Tessler, J. Arroyave, S. Contreras and A. Oce-
guera-Figueroa (CNI 25); Bosque Azul, Chiapas (16°07'37.83"N, 91°43'53.68"W; 1462 m) 3 speci¬
mens collected January 25, 2016 by M. Tessler, J. Arroyave, S. Contreras and A. Oceguera-Figueroa
(CNI 26). GenBank coxl barcode sequence accessions: MG821608, MG821609, and MG821610.

Helobdella octatestisaca Lai and Chang, 2009
Figures 14-15

The presence of a chitinous nuchal scute; dorsal and ventral surfaces with inconspicuous
papillae; no metameric pigmentation patterns; eyespots in one pair, each shaped somewhat tri¬
angularly, almost touching each other is consistent with the description of Helobdella octatestisaca
(see Salas-Montiel et al., 2014). Found on the underside of submerged rocks and plants. Non¬
blood-feeding (liquidosomatophagous). New state record; new locality records: Bosque Azul,
Lagunas de Montebello (16°07'37.83"N, 91°43'53.68"W; 1462 m) 30 specimens collected January
25, 2016, by M. Tessler, J. Arroyave, S. Contreras, and A. Oceguera-Figueroa (CNI 27); Osuma-
cinta Dam (16°57'47.5"N, 93°06'21.82"W; 207 m) 16 specimens collected January 24, 2016, by
M. Tessler, J. Arroyave, S. Contreras, and A. Oceguera-Figueroa (CNI 28). GenBank coxl barcode
sequence accessions: MG821611, MG821612, MG821613, and MG821614.

Helobdella socimulcensis Caballero, 1931
Figures 16-17

The presence of dorsal surface with complex pattern of longitudinal stripes and metameric
papillae; nuchal scute absent; eyespots in one pair, punctiform, well separated from each other is
consistent with the description of Helobdella socimulcensis (see Salas-Montiel et al., 2014). Found
on the underside of submerged rocks and plants. Non-blood-feeding (liquidosomatophagous).
New locality records: Rio Fogotico, Arcotete, Chiapas, Mexico (16°44'46.5"N, 92°34'17.24"W;
2298 m), collected January 23, 2016, by M. Tessler, J. Arroyave, S. Contreras, and A. Oceguera-
Figueroa (CNI 29). GenBank coxl barcode sequence accessions: MG821615 and MG821616.

Placobdella ringueleti Lopez-Jimenez and Oceguera-Figueroa, 2009

Figures 18-19

The presence of two pairs of eyespots in close contact, a single dorsal longitudinal stripe,
and a papillated dorsal surface is consistent with the description of Placobdella ringueleti. New
locality record: Palenque (17°29'10"N, 92°01'10"W; 68 m), collected March, 2015, by J. Perez-
Flores (CNHE 10455). GenBank coxl barcode sequence accession: MG821617.

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FIGURES 16-19. Rhynchobdellida from Chiapas, Mexico. Helobdella socimulcensis : 16 . dorsal view, 17 . ventral
view. Placobdella ringueleti: 18 . dorsal view, 19 . ventral view.

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CONCLUSIONS

With the description of Erpobdella adani there are now 33 known leech species from Mex¬
ico (Oceguera-Figueroa and Leon-Regagnon, 2014; Salas-Montiel et al., 2014). Accordingly,
the nine species found in Chiapas (six non-blood-feeding and three blood-feeding) represent
27% of the leeches found in Mexico. Four of the 10 leech families known from Mexico (40%)
were found as well. The diversity found thus far in Chiapas is similar to that known from the
relatively well-surveyed central states of Mexico. However, the diversity of the state is surely
higher than presently known. For example, the marine species of the Piscicolidae and Ozo-
branchidae have yet to be surveyed in the area, as far as we are aware.

All but two leech species found are aquatic. The terrestrial leech Diestecostoma octannulata
is previously known only from Volcan Tajumulco, Guatemala (Moore, 1946), and thus the
record reported here expands the known distribution of this species to Mexico. Another ter¬
restrial leech is potentially known for the state: D. magna Moore, 1945 (Oceguera-Figueroa
and Leon-Regagnon, 2014); however, the type locality of this species is ambiguous, being
between the states of Veracruz and Chiapas (Moore, 1945). Two Helobdella species were found
in addition to the previous record of H. socimulcensis —these three species are relatively wide¬
spread in Mexico and correspond to half of the known Mexican Helobdella species (Salas-
Montiel et al., 2014). However, H. elongata from Chiapas might be a new taxon given the
elevated genetic distances in comparison with samples from other localities in Mexico. The
three blood-feeding species, Pintobdella chiapasensis, Placobdella ringueleti, and Haementeria
acuecueyetzin were found in prior works (Caballero, 1957; Lopez-Jimenez and Oceguera-
Figueroa, 2009; Phillips et al., 2010; Perez-Flores et al., 2016).

The addition of the new species, Erpobdella adani, along with current and prior records of
E. triannulata represent half of the four Erpobdella species now known from Mexico (Oce¬
guera-Figueroa and Leon-Regagnon, 2014). Erpobdella adani is phylogenetically distinct from
those Erpobdella species included in the analysis. It is generally closely related to the other three
species known from Mexico, as well as most of the North American Erpobdella. Of the three
other Mexican species, E. adani is most closely related to E. ochoterenai. The relationships
recovered for Mexican Erpobdella in our analyses are largely consistent with prior work (Oce¬
guera-Figueroa et al., 2005, 2011), insofar as the positions of E. mexicana and E. ochoterenai
are consistent while E. triannulata varies in its position depending on the analysis.

Unlike the other Mexican species that are broadly distributed (Oceguera-Figueroa and
Leon-Regagnon, 2014), Erpobdella adani was found only in a single stream. Future collecting
efforts for this species should concentrate on more streams in Chiapas and possibly in nearby
Guatemala, which, like much of Central America, remains largely unsurveyed for leech diver¬
sity. Until then, it will be hard to determine how restricted the range is of this putative endemic.
It is also worth noting that leech-collecting efforts generally focus on lacustrine habitats, while
E. adani was found in lotic conditions. Furthermore, these conditions made it generally hard
to spot and collect.

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ACKNOWLEDGMENTS

We would like to thank the AMNH Roosevelt Grant for funding fieldwork for M.T. and
the PAPIIT-IA202016 and IA204114 projects awarded to A.O.-F. for funding fieldwork and
laboratory expenses; Ofelia Delgado, Gisela Martinez Flores, Andrea Jimenez Marin, and Laura
Marquez for providing assistance in the molecular lab; Jairo Arroyave, Samantha Contreras,
Hector Tejero, and Iris Mendez for help collecting leech specimens; Luis Garcia Prieto and Lily
Berniker for support in the collection management; and Susana Gomez for aiding with the
preparation of pictures.

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