Corresponding author: Daniel Fernández Marchán (
Academic editor: R. Blakemore
The earthworm family Hormogastridae shows a remarkable disjunction in its distribution in the Iberian Peninsula, with the
Marchán DF, Fernández R, Novo M, Cosín DJD (2014) New light into the hormogastrid riddle: morphological and molecular description of
The increasing availability of molecular and ecological data has placed the integrative taxonomy (as defined by
Taxonomic characters traditionally used for the study of earthworms are few and sometimes present high intraspecific variability (
On the other hand, it seems that most key characters used for hormogastrid traditional taxonomy and phylogeny (notably the shape, number and position of the spermathecae) are highly homoplasic, showing little or no phylogenetic signal across the family.
Due to its relevance for this subject, the intermediate area between the main ranges of hormogastrids in Spain has been subject to recent sampling campaigns. Both Zaragoza and Teruel (Aragón, Spain) were suitable regions as they have been poorly sampled for earthworms unlike the surrounding provinces. While no success was met in Zaragoza, a population assignable to a new species of
This paper focuses on the description of
Specimens were collected by hand and fixed in the field in ca. 96% EtOH, with subsequent alcohol changes. Once in the laboratory, specimens were preserved at -20 °C.
The studied material includes 10 specimens (five mature specimens, one semimature specimen with tubercula pubertatis and four immatures) collected in a cleared holm-oak wood at the foothill of Sierra de Oriche, road A-2514 between Huesa del Común and Rudilla, Teruel (Spain) (
Map of the Iberian Peninsula showing the collection site of
Specimens have been deposited in the Oligochaete collection of the Departamento de Zoología y Antropología Física, Universidad Complutense de Madrid (UCMLT), Spain with vouchers UCMLT 00001-00010.
Specimens available from previous studies (
Total genomic DNA was extracted from ventral integument tissue samples using the DNeasy Tissue Kit (QIAGEN) with two consecutive steps of elution (70 µl of buffer). Seven molecular regions were amplified: mitochondrial subunit 1 of cytochrome
Holo- and paragenetypes (sensu
Specimen | Voucher | COI | 16S-tRNAs | 28S rRNA | H3 |
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HRUD1 | UCMLT 00001 |
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HRUD2 | UCMLT 00002 |
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HRUD4 | UCMLT 00004 |
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HRUD5 | UCMLT 00005 |
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HRUD6 | UCMLT 00006 |
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HRUD7 | UCMLT 00007 |
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HRUD8 | UCMLT 00008 |
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HRUD9 | UCMLT 00009 |
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HRUD10 | UCMLT 00010 |
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The new sequences were combined with all the hormogastrid information available from previous studies (
Sequences of each individual gene were aligned in MAFFT (
Bayesian Inference (BI) of the phylogeny was estimated with MRBAYES v.3.1.2 (
Uncorrected pairwise differences for the mitochondrial regions were calculated between
Holotype. Adult (UCMLT 00003),
(
Length of mature specimens*: 178–180 mm.
Maximum diameter (pre-clitellar, clitellar, post-clitellar) of mature specimens: 8–10, 9–11, 7–10 mm.
Number of segments*: 305–369.
Weight (fixed specimens)*: 7.05–11.57 g.
Colour: From light brown to dark chocolate brown varying between individuals, with orangeish-brown clitellum of a lighter shade on living specimens (
Prostomium prolobic, longitudinal striation on segments 1 and 2.
Closely paired chaetae; interchaetal ratio at segment 40,
Spermathecal pores at intersegments 9/10 and 10/11 at the level of
Male pores open over chaetae
Clitellum saddle-shaped extending over segments (13) 14–28. Tubercula pubertatis on 1/n 22-27(1/n 28) as a continuous line. Papillae of chaetae ab in variable positions, usually between segments 12 and 28: papillae on 12 always showing an unusual degree of development in mature individuals, being very conspicuous both in live and fixed specimens (
Funnel shaped, strongly thickened septa in 6/7, 7/8 and 8/9, septum 9/10 slightly thickened. The latter’s attachment to the dorsal body wall is displaced two segments backwards, creating a mismatch between inner and outer segmentation with an internally very wide segment 9.
Last pair of hearts in segment 11. Three shiny, strongly muscular gizzards in 6, 7 and 8. Not apparent Morren’s glands, even though small wrinkles exist in the oesophageal wall between segments 10 and 16.
A posterior gizzard is not well differentiated. There is a slight dilatation of the oesophagus between 14 and 16, but it lacks the muscular wall and reinforcements of a true gizzard. First section of the intestine is not dilated.
Typhlosole begins around segments 20 and 21 with seven lamellae, which around segments 26–27 increase to nine. From there they decrease gradually in number until segments 80–105, where they fuse in a single lamella. The latter extends until segments 218-230, where the typhlosole ends.
Fraying testes and iridescent seminal funnels in 10 and 11. Two pairs of voluminous, grainy seminal vesicles in 11 and 12. Ovaries and female funnels in 13, ovisacs in 14.
Two pairs of spermathecae in intersegments 9/10 and 10/11 (but apparently contained in segment 9 due to septum’s backward displacement), the posterior pair bigger. They are sessile and disc-shaped, with multiple inner chambers which open to the exterior through a common pore, in the intersegments 9/10 and 10/11. Some individuals show double spermathecae (each multicameral and with own pore), either in 9/10 or 10/11 (
Anterior nephridial bladders U-shaped with very close branches and no apparent cecum (
Known only from its type locality.
The specimens were collected at 10–20 cm deep in the soil in a cleared holm-oak wood, at the border between a dense forest of
The species is named after Jose Antonio Fernández Fernández, father of the first author Daniel Fernández Marchán and important contributor during the sampling campaign in which this species was discovered.
Analyses were conducted on sequences from loci COI (10 individuals), 16S (2 individuals), 28S (2 individuals) and H3 (2 individuals) of the new species, combined with similar sequences from other hormogastrid species.
The resulting Bayesian inference of the phylogenetic tree is shown in
Bayesian inference of the phylogenetic tree on the concatenated sequence. Numbers above branches indicate posterior probability/bootstrap (of the Maximum Likelihood analysis) support values higher than 0.9/70 (shown as asterisks on terminal branches). Black rectangles show clades not recovered in both analyses (the alternative is shown with a dashed line). The cryptic species included in
Uncorrected pairwise distances for the genes COI and 16S-tRNA for
Uncorrected pairwise distances for the genes COI (below the diagonal) and 16S-tRNA (above the diagonal) for
HJOS | HE3 | HE1 | HE2 | HE5 | HE4 | XAN | HPRE | HNAJ | HEM* | HEM** | |
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0.14/0 | 13.10 | 14.20 | 12.50 | 19.41 | 13.50 | 14.23 | 14.28 | 15.31 | 17.40 | 16.07 |
HE3 | 18.10 | 0.29/0 | 9.87 | 9.96 | 17.18 | 12.34 | 14.37 | 15.93 | 16.69 | 17.54 | 15.57 |
HE1 | 17.77 | 15.51 | 10.03/4.10 | 7.97 | 17.83 | 12.95 | 15.54 | 17.73 | 17.54 | 17.26 | 16.56 |
HE2 | 16.47 | 14.16 | 15.13 | 1.75/0.67 | 17.03 | 13.38 | 14.93 | 16.62 | 18.18 | 16.70 | 16.70 |
HE5 | 16.83 | 16.28 | 17.48 | 16.36 | 0.34/0 | 16.37 | 21.04 | 21.55 | 22.37 | 22.28 | 21.32 |
HE4 | 19.08 | 15.67 | 17.37 | 16.86 | 10.38 | 3.75/1.75 | 15.49 | 18.06 | 17.51 | 17.81 | 16.53 |
XAN | 18.30 | 18.26 | 18.36 | 18.96 | 17.01 | 18.49 | 0.37/0.19 | 11.60 | 13.58 | 14.34 | 12.66 |
HPRE | 18.61 | 20.17 | 20.34 | 19.74 | 18.92 | 19.52 | 17.76 | 0/2.14 | 10.74 | 16.47 | 13.69 |
HNAJ | 18.92 | 18.39 | 19.77 | 18.19 | 18.64 | 19.17 | 19.92 | 17.31 | 0.10/0.18 | 16.69 | 14.86 |
HEM* | 18.38 | 18.52 | 19.17 | 20.45 | 17.06 | 18.58 | 20.45 | 19.67 | 19.92 | 3.50/1.97 | 8.76 |
HEM** | 18.11 | 18.19 | 18.10 | 17.79 | 16.14 | 16.55 | 18.31 | 19.24 | 18.93 | 17.63 | 6.30/2.07 |
Both morphological and molecular characters of
The species
Comparison of the morphological characters of
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Colour | Brownish | Colourless | Colourless | Slightly greyish | Dark brownish | Brownish grey |
Clitellum | (13)14–28 | (13)14(15)–26(27)28 | 14–26 | 13–31 | 13,14,17–27,28 | 1/14,15–29,1/2 30 |
Tubercula pubertatis | 1/n 22–27 (1/n 28) | 22(23)–25(26) | 23–26 | 20–26 | (20)21–27 | 22–28 |
Length (mm) | 178–180 | 92–200 | 20–161 | 188–230 | 154 | 200–325 |
N. segments | 305–369 | 205–300 | 190–230 | 395–523 | 243–278 | 320–429 |
Weight (g) | 7.05–11.57 | 1.96–9.67 | 0.59–4.23 | 22.6–31.4 | 6.57 | 12.85–29.38 |
Spermathecae position (pores) and appearance | 9 (see text) (9/10,10/11) Simple(double) Multicameral, disc shaped | 9,10 (9/10,10/11) Simple Tubular | 10,11 (9/10,10/11) Simple Small, globular | 10,11 (10/11,11/12) Multiple Small, globular | 9,10 (9/10,10/11) Simple Multicameral, disc shaped | 9,10 (9/10,10/11) Simple Globular |
N. typhlosole lamellae | 9 | 5 | 12 | 15–17 | 15 | 21–23 |
Thickened septa | 6/7,7/8,8/9, (9/10) | 6/7,7/8,8/9, (9/10) | (6/7),7/8,8/9, 9/10,(10/11) | 6/7,7/8,8/9, (9/10) | 7/8,8/9,9/10, (10/11) | 7/8,8/9,9/10, (10/11) |
Other hormogastrid species possess double or multiple spermathecae, but never of the multicameral, disc shaped kind.
The geographically closest species,
The
Based on their phylogenetic and morphological relatedness, an origin of
While
Based on its distinctive morphology and geographic range, high genetic divergence and consistent recovery as a well-defined clade,
At this stage it is more conservative to assign
We are indebted to Jose Antonio Fernández Fernández for his help during the sampling campaign. Subject Editor Rob Blakemore and two anonymous reviewers helped to improve the manuscript. This research was funded by project CGL2010-16032 from the Spanish Government.
GenBank accession numbers for all sequences used in the phylogenetic analysis, including outgroups. RF: sequences provided by Rosa Fernández.
Species | COI | 16S-tRNAs | 28S-rRNA | H3 |
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RF |
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