Zoosyst. Evol. 100 (1) 2024, 155-166 | DOI 10.3897/zse.100.118675

ye Te
BERLIN

Updated taxonomy and new insights into the evolutionary relationships
of the genus Sporonchulus Cobb, 1917 (Nematoda, Mononchida) after
the study of two Vietnamese species

Tam T. T. Vul*, Anh D. Nguyen, Thi Mai Linh Le, Reyes Pefia-Santiago*

1 Institute of Ecological and Biological Resources, Vietnam Academy of Sience and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
2 Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
3 Departamento de Biologia Animal, Biologia Vegetal y Ecologia, Universidad de Jaén, Campus ‘“‘Las Lagunillas”’ s/n, Edificio B3, 23071- Jaén, Spain

https://zoobank. org/DA6152E1-3DDF-402B-999F-E4024F916D36

Corresponding author: Tam T. T. Vu (vtam7572@gmail.com)

Academic editor: Pavel Stoev # Received 12 January 2024 Accepted 6 February 2024 Published 15 February 2024

Abstract

Two known species of the genus Sporonchulus, namely S. ibitiensis and S. vagabundus, collected from natural areas of Vietnam,
are characterized, including descriptions and illustrations of both species, as well as SEM observations and molecular (18S-, 28S
rDNA) analyses of S. ibitiensis. The identity of the two species is discussed, with detailed comparison with previously known popu-
lations. The taxonomy of the genus is updated, presenting a diagnosis, list of species, key to their identification, and a compendium
of their main morphometrics. An integrative analysis, combining morphological data with a cladistic approach and the first molec-
ular study for a representative of Sporonchulus, better supports a narrow relationship of this genus with Mononchidae than with

Mylonchulidae members, however further research should be conducted to elucidate its phylogeny.

Key Words

Description, molecular analysis, morphology, phylogeny, 18S, 28S-rDNA

Introduction

Mononchs, the members of the nematode order Monon-
chida, are an interesting taxon due to their wide geo-
graphical distribution and their role as active predators.
Their 47 valid genera and 432 valid species (Hodda 2022)
dwell in all kind of continental, both soil and freshwater,
habitats in the six continents, even Antarctica. They are
comparatively large nematodes, often ranging from | to
3 mm long, show a very active predatory behaviour to-
ward other nematodes, including cannibalism, and play
an important role in feeding relationships of nematode
assemblages. Ahmad and Jairajpuri (2010) provided an
excellent monograph devoted to their morphology and
taxonomy, which still is the best reference for a general
overview of the group.

The genus Sporonchulus is a rare mononchid taxon,
with only four valid species, that, however, display a
Pantropical distribution, recorded in Neotropics, Africa
and Indomalayan region (Ahmad and Jairajpuri 2010).
Originally proposed by Cobb (1917) as a subgenus of
Mononchus Bastian, 1865 with M. (S.) dentatus Cobb,
1917 as its type and only species, its rank was raised to
genus level by Andrassy (1958). Taxonomy of Sporon-
chulus was studied by different authors (Mulvey 1963;
Jairajpuri 1971; Andrassy 1993; Ahmad and Jairajpuri
2010) and its position in Mononchida tree was a matter
of some controversy. Thus, Jairajpuri (1969) created the
subfamily Sporonchulinae, within the family Mylonchu-
lidae Jairajpuri, 1969, to include the genera Granonchu-
lus Andrassy, 1958, Judonchulus Andrassy, 1958, Prion-
chuloides Mulvey, 1963 and Sporonchulus Cobb, 1917.

Copyright Vu, T.T.T. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited.

156 Vu, T.T.T. et al.: The new insights into the evolutionary relationships of the genus Sporonchulus

This idea was followed in subsequent major contributions
co-authored by the same author (Jairajpuri and Khan
1982; Ahmad and Jairaypuri 2010). Nevertheless, other
authors (Andrassy 1976, 1993, 2009; Zullini and Peneva
2006) did not accept Jairaypuri’s scheme, did not recog-
nise Sporonchulinae as a valid taxon, and considered all
its genera as members of Mononchinae Filipjev, 1934
in Mononchidae Filipjev, 1934. These authors provided
morphological evidence in favour of their proposals, but
no molecular analyses were available to support them.
Besides, Loof (2006) stated (p. 308) that “this genus is in
need of revision”.

The occurrence of Sporonchulus in Vietnam is not re-
ported so far. Nevertheless, a population of Sporonchulus
ibitiensis (Carvalho, 1951) Andrassy, 1958 was collected
in the course of a general nematological survey conduct-
ed to explore the mononchid diversity of natural enclaves
of the country, and fresh specimens were therefore avail-
able for sequencing. Besides, the revision of the materi-
al previously identified as Actus conoidus Dhanachand,
Romoni & Pramodini, 2006 by Vu (2017) has revealed
that it belongs to S. vagabundus Jairajpuri, 1971. Thus,
this contribution aims to provide a morphological charac-
terisation of the Vietnamese specimens of the mentioned
above species, to carry out the first molecular study of a
representative of the genus, to discuss their evolutionary
relationships and to provide its updated taxonomy.

Materials and methods

Sampling, extraction and processing of
nematodes

Three females of S. vagabundus, previously deposited
at Department of Nematology, Institute of Ecology and
Biological Resources (IBSR), Vietnamese Academy of
Sciences and Technology (VAST), Hanoi, Vietnam, were
available for study. Four females of S. ibitiensis obtained
from soil samples were collected in a natural forest at
Tram Tau town, Yen Bai Province and Du Gia Natural
Reserve, Ha Giang Province (Vietnam). Nematodes
were extracted using a modified Baermann funnel tech-
nique (Southey 1986), heat killed, fixed in TAF solution
(Southey 1986) for morphological observations or in a
DESS mixture (Yoder et al. 2006) for molecular analyses.
Then, specimens were transferred to anhydrous glycerol
(Seinhorst 1959, 1962), and mounted on glass slides for
their observation with light microscopy. Nematodes were
measured and photographed using an Eclipse 801 micro-
scope (Nikon, Tokyo, Japan) with differential interference
contrast optics, a drawing tube (camera lucida) and a DS
digital camera. Line drawings were made from taken pho-
tomicrographs, after processing with Adobe Photoshop
CS8. Morphometrics include Demanian indices and other
measurements and ratios, some of them presented in a sep-
arate table; meanwhile, others form part of the literal de-
scription of the species. All measurements were recorded

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in um, except body length in mm. After filming and taking
pictures, selected specimens were submitted for molec-
ular studies. SEM photographs were taken using SEM
PRISMA E according to Abolafia (2015) at VAST. Pic-
tures were edited using Adobe Photoshop CC2018.

DNA extraction, polymerase chain reaction
(PCR) and sequencing

Nematode DNA of S. ibitiensis was extracted from a sin-
gle individual as described by Holterman et al. (2006) and
DNA extracts were stored at —20° until used as PCR tem-
plate. The D2-D3 expansion segment of 28S rDNA and
18S were amplified using the forward D2A (5'-ACAAG-
TACCGTGGGGAAAGTTG-3’) and reverse D3B (5'—
TCGG AAGGAACCAGCTACTA-3') primers (Subbotin
et al. 2006) and primers 18S (18F: 5’°-TCTAGAGCTA-
ATACATGCAC-37/18R: 5’-TACGGAAACCTTGT-
TACGAC-3’). All PCR reactions contained 12,5 wl Hot
start green PCR Master Mix (2x) (Promega, USA), 1 ul
of the forward and reverse primer (10 uM each), the 3 ul
DNA template and sterile Milli-Q water to 25 ul of the
total volume. All PCR reactions were performed in Sim-
pliAmp Thermal cycler (Thermo Fisher scientific) as
follows: an initial denaturation step at 95 °C for 4 min,
followed by 40 cycles at 95 °C for 30 s, 54 °C for 30 s and
72 °C for 60s with a final incubation for 5 min at 72 °C.
Amplicons were visualised under UV illumination after
Simply safe gel staining and gel electrophoresis. Purified
PCR products were sent to Apical Scientific Company
for sequencing (Selangor, Malaysia). After sequencing
the obtained rDNA sequences fragments were deposit-
ed in GenBank under the following accession numbers:
0Q377123 (18S) and 0Q377128 (28S).

Phylogenetic analyses

For exploring phylogenetic relationships, analyses were
based on 18S and 28S rDNA. The newly obtained se-
quences were manually edited using Chromas 2.6.6
(Technelysium, Queensland, 110 Australia) and aligned
with other sequences available in GenBank using Clust-
alW alignment tool implemented in the MEGA11 (Kumar
et al. 2021). Poorly aligned regions at extremes were re-
moved from the alignments using MEGA7. The best fit
model of nucleotide substitution used for the phyloge-
netic analysis was statistically selected using jModelTest
2.1.10 (Darriba et al. 2012). The phylogenetic tree was
generated with the Bayesian inference method using Mr-
Bayes 3.2.6 (Ronquist et al. 2012). The analysis under the
generalised time reversible and invariant sites and gam-
ma distribution (GTR + I + G) model was initiated with
a random starting tree and run with the Markov chain
Monte Carlo (Larget and Simon 1999) for 1 x 10° gen-
erations. The tree was visualised and saved with FigTree
1.4.4 (Rambaut 2018).

Zoosyst. Evol. 100 (1) 2024, 155-166

Results

Descriptions of species

Sporonchulus ibitiensis (Carvalho, 1951) Andrassy, 1958

Mononchus ibitiensis Carvalho, 1951. Syn.
Sporonchuloides ibitiensis (Carvalho, 1951) Mohandas & Prabhoo, 1982.

Material examined. Four females from one location, in
good state of preservation.

Morphometrics. See Table 1.

Description. Female. Moderately slender to slen-
der (a = 28-34) nematodes of medium size, 1.09—1.37
mm long. Body cylindrical, slightly tapering towards the
anterior end and more appreciably towards the posteri-
or extremity as the tail is conical. Upon fixation, habitus
strongly curved ventrad, C- to G-shaped. Cuticle smooth
when observed with LM, but showing very fine transverse
striation under SEM, two-layered, | um thick at anterior
region, 1.5 um in midbody and 1.5—2 um on tail. Lip re-
gion almost continuous with the adjoining body, 2.0—2.2
times as wide as high and one-half to two-thirds (53-—
67%) of body diameter at neck base, with totally fused
lips and weakly protruding papillae; SEM observations
(Fig. 1D): oral field comparatively small, with almost
hexagonal oral aperture surrounded by six perioral liplets,
labial papillae button-like, prominent, cephalic papillae
also button-like, but visibly smaller than labial ones. Am-
phid fovea small, goblet-like, located at 10-11 um from
the anterior end, its aperture a short transverse slit 3—3.5
um long, occupying up to one sixth (12-16%) of lip re-
gion diameter. Vestibulum 4.5—5.5 um long. Buccal cav-
ity up to twice (1.8—1.9 times) as long as wide, 1.1—1.3
times longer than lip region diameter: vertical (anterior)
plates somewhat convergent at their anterior and posterior
ends, their walls 1-1.5 um thick, horizontal (posterior or
basal) plates visibly oblique, with foramina, dorsal tooth
apex situated at anterior third of buccal cavity (68-74%
from the base), anterior subventral plates bearing each
two irregular rows of small teeth with decreasing size
from the base till the level of dorsal tooth. Anterior end of
pharynx embracing the basal part of buccal cavity, gland
nuclei obscure except S,N = 81-85%. Nerve ring locat-
ed at 92-101 um or ca one-third (31-36%) of the total
neck length. Pharyngo-intestinal junction non-tubercu-
late, 14-18 =< 8.5—10 um. Genital system diovarian, with
small and equally developed branches occupying 6—13%
of body length: ovaries comparatively large, 50-146 um
long, with oocytes first arranged in several rows and then
in only one row; genital tract very short and poorly dif-
ferentiated, oviduct 35-48 um long or 1.0—1.2 body di-
ameters, pars dilatata oviductus not enlarged, uterus
17-33 um long or 0.5—0.7 body diameters, both separat-
ed by an indistinct weak sphincter; vagina 11 um long,
extending inwards to one-third (34%) of body diameter,
pars proximalis 6.5 x 1.5 um, pars refringens with two
drop-shaped or somewhat trapezoidal sclerotized pieces

157

Table 1. Main morphometrics of two Sporonchulus species
found in Vietnam. Measurements in um except L in mm.

Species S. ibitiensis S. vagabundus
n 4°92 329
Character
L 1.26 + 0.12 (1.09-1.37) 1.27-1.41
a 29.9 + 2.8 (28-34) 34-39
b 4.1 + 0.2 (3.8-4.2) 4.6-4.8
c 24.0 + 1.5 (22-25) 23-25
e 2.2 + 0.0 (2.1-2.2) 2.4-2.5
V 60.4 + 0.9 (60-62) 57-59
Lip region diameter 24.1 + 1.9 (21-26) 20-21,
Buccal cavity length 27.3 + 0.5 (27-28) 22-23
Buccal cavity width 14.5 +1.7 (12-16) 11.5-12.5
Dorsal tooth apex (%) 71.6 + 2.8 (68-74) 73-75
Neck length 293 + 13 (284-302) 278-293
Body diameter at neck base 35.5 + 4.9 (32-39) 34-36
midbody 42.8 +7.3 (32-48) 33-40
anus 24.8 + 3.3 (21-29) 22-24
Distance vulva — ant. end 761 + 64 (674-824) 748-806
Rectum length 21.0 + 3.8 (16-24) 20-23
Tail length 52.8 + 6.7 (46-62) 53-60

2.5 x 2.5 um and a combined width of 5 um, pars distalis
1.5 um, vulva a transverse slit. Rectum 0.8—1.0 times the
anal body diameter long. Tail conical with finely round-
ed tip, regularly curved ventrad, with poorly developed
glands and lacking a terminal spinneret (Figs 1, 2).

Male. Not found.

Molecular characterization. After sequencing and
editing, two sequences were obtained for phylogenetic
analyses: one full length 18S rDNA with 1591 bp length
(GenBank accession N° OQ377123) and one_near-
ly 745 bp length D2D3 of LSU rRNA (28S) (GenBank
accession OQ377128).

Locality and habitat. Vietnam, Yen Bai Province,
Tram Tau town (coordinates 21°50'18"N, 104°44'22"E,
altitude 930 m) and Ha Giang Province, Du Gia Natural
Reserve, (coordinates 22°43'18"N, 105°11'38"E, altitude
780 m) where the nematodes were found in soil around
the roots of forest trees.

Voucher specimens. Permanent slides are stored at
the Department of Nematology, Institute of Ecology and
Biological Resources, VAST, Hanoi, Vietnam.

Remarks. Present description provides new data and
illustrations of S. ibitiensis, especially useful for com-
parative purposes. General morphology of Vietnamese
females very well fits that of type specimens and other
known populations (Mulvey 1963; Mulvey and Jensen
1967; Lordello 1970; Chaves and Geraert 1977; Mohan-
das and Prabhoo 1979; Jairaypuri and Khan 1982; Chaves
1990; Loof 2006; Tahseen et al. 2013; Perichi et al. 2021)
of the species. Nevertheless, their morphometrics need
further analysis. Table 1 shows the most relevant mea-
surements and ratios of females herein studied; mean-
while, Table 2 includes those available from the literature.
Especially relevant is the variation observed in buccal
cavity length (18-34 um), an unusually wide range in
mononchid species. Actually, Vietnamese specimens dis-
play 26—28 um long buccal cavity, totally comparable or

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158

C /

Vu, T.T.T. et al.: The new insights into the evolutionary relationships of the genus Sporonchulus

Ny

Figure 1. Sporonchulus ibitiensis (Carvalho, 1951) Andrassy, 1958 (Female). A. Entire; B—D. Anterior body region, lateral median
(B, C) and lateral submedian (D) view; E. Neck region; F. Anterior genital branch; G. Anterior body region, lateral surface view;
H. Caudal region; I. Pharyngo-intestinal junction; J. Vagina. Scale bars: 200 um (A); 10 um (B—D, F, H, I); 50 um (E); 5 um (G, J).

with appreciably overlapping ranges to those reported for
Afrotropical (Mulvey and Jensen 1967; Chaves and Ger-
aert 1977) and Indomalayan (Mulvey 1963; Mohandas
and Prabhoo 1979; Jairajpuri and Khan 1982; Loof 2006)
populations, but they differ from some South American
specimens (Carvalho 1951; Chaves 1990), 25-33 vs 18—
23 um, indeed a remarkable difference that might be the
result of a biogeographical pattern with two tentative spe-
cies or subspecies. Nevertheless, a doubt persists over the
true identity of several of these populations, which should
be resolved before proposing any taxonomical change.
Thus, South American females recorded by Carvalho

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(1951) and Chaves (1990) were not characterised enough
for a comparative analysis. The two Brazilian females
studied by Lordello (1970) showed 23 and 27.5 um long
buccal cavity. Chaves and Geraert (1977) studied three
females from the former Zaire characterised by bearing
a terminal subdorsal pore, which might be a remarkable
distinguishing trait. Besides, two (more recent) contribu-
tions (Tahseen et al. 2013; Perichi et al. 2021) raise more
uncertainties. On the one hand, Tahseen et al. (2013)
studied only one Indian female with 22 um long buc-
cal cavity, which suggests that the species might display
more variation in Indomalayan range than previously re-

Zoosyst. Evol. 100 (1) 2024, 155-166

159

Figure 2. Sporonchulus ibitiensis (Carvalho, 1951) Andrassy, 1958 (Female, LM). A. Entire; B, C, F, G. Anterior body region, later-
al median (B, C) and lateral submedian (F, G) view; C. Lip region (SEM), in face view; E. Neck region; H. Caudal region. I. Vagina;
J. Pharyngo-intestinal junction. Scale bars: 200 um (A); 10 um (B, C, F, G, H, J); 5 um (D, I); 50 um (E).

ported. On the other hand, Perichi et al. (2021) described
two Venezuelan populations whose buccal cavities dis-
play very wide variation in their length (27—33 and 24—34
um) and with appreciable difference in their lip region
width (26—30 and 20—27 um, respectively).

Sporonchulus vagabundus Jairajpuri, 1971

Material examined. Three females from one location, in
good state of preservation.

Morphometrics. See Table 1.

Description. Female. Moderately slender to slender
(a = 28-35) of medium size, 1.27—1.41 mm long. Body
cylindrical, slightly tapering towards the anterior end and
more appreciably towards the posterior extremity as the
tail 1s conical. Upon fixation, habitus strongly curved ven-

trad, C- to G-shaped. Cuticle smooth, two-layered, 1 um
thick at anterior region, 1—1.5 um in midbody and 2 um
on tail. Lip region almost continuous with the adjoining
body, 2.1—2.5 times as wide as high and hardly more than
one-half (53%) of body diameter at neck base, with to-
tally fused lips and weakly protruding papillae. Amphid
fovea small, goblet-like, located at 8—10 um from the an-
terior end, its aperture a short transverse slit 3 um long,
occupying less than one-sixth (14-15%) of lip region di-
ameter. Vestibulum 5 um long. Buccal cavity up to twice
(1.8—1.9 times) as long as wide, hardly longer (1.1 times)
than lip region diameter: vertical (anterior) plates some-
what convergent at their anterior and posterior ends, their
walls 1—1.5 um thick, horizontal (posterior or basal) plates
visibly oblique, with foramina, dorsal tooth apex situated
at three-fourths of buccal cavity (74-75% from the base),
anterior subventral plates bearing each two irregular rows

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160

Vu, T.T.T. et al.: The new insights into the evolutionary relationships of the genus Sporonchulus

Table 2. Compilation of main morphometrics of Sporonchulus species. Measurements in um except L in mm.

Character

Species
S. coronatus
S. dentatus

?
?
S. Ibitiensis

S. vagabundus
?
?

n L

399 1.07-1.40
2 iba

oe Tes
Q 1.25

2 1141.15
092 11415
9 1.4

9 1.2

599 «11-14
399 1.01-+1.05
292 0,99, 1.37
2199 1.24-1.43
43h 1.18+1.28
829 0.95-1.12
3022 1.26-1.73
Q 1.10

11992 1.01.5
499 1,09-1.37
3992 1.26-1.34
O92 12-18
10292 0.91-1.09
300) 127-1.

a

b

Cc

V

17-22 3.3-4.0 20-26 60-66

oa
30-31*
30
26-27
26-30
24
30
28-36
23-25
21, 26
20-28
27-30
25-32
24-34
27
22-33
28-34
28-36
22-32
20-29
34-39

oye
4.0*
3.9
34
3.4-3.9
4
3.9
3,9-4.3
3.0
3.4, 3.9
3.9-4.4
3.8-4.0
3.5-4.2
3.8-5.0
4.0
3.4-5.2
3.8-4.2
4.2-4.6
3.7-4.8
3.6-4.2
4.6-4.8

20*
18-19"
19
21-23
19-23
23:
24
21-23
17-21
20, 26
23-28
23-24
17-19
20-27
20
17-28
22-25
22-25
21-27
19-22
23-25

62
61
62
62-64
62-64
60
63
57-63
57-60
7261
59-63
58-60
58-64
61
59-63
60-62
58-61
51-63
59-64
57-59

Cc

y
2.6,
ra

E
a

2.0-2.5
Pe
26°

?
aN

?

1.7-2.1
1.6-1.7
2.1-2.7
1.8-3.1
2.0
1.4-2.3
2.1-2.2
2.0
20-25
1.7-2.4
2.4-2.5

Ird bel neck abd tail spic vm country Ref.!
z 28 ? i ? - - Brazil TZ
20% SS, 297* Ze 55° - ~ Brazil 3
25-30 31-36 272-231* 22-26* 60-71 - Mauritius 4
? 34 po 7 ? 65 - - Venezuela 2
? 32-34 330-335 ? 49-55 - - Brazil 5
2? 32-35 ? ? ? - ~ Several 6
23° AS" 350* 24 60 - - Brazil 7
? 25 308* 19* 50 - - Srilanka 2
? = 27-28 ? ? 52-62 - - Nigeria 8
25* 26 ? 20* 47* - - Zaire 9
2? 23,27 291, 344 4 49,53 - - Brazil 5
22-28 28-30 . a 47-60 - - India =-'10, 11
22-28 30-31 . a 52-54 30-35 11-12
19-23 21-23 210-237 ? 49-64. - - Argentina 12
20-28 26-33 299-396 21-33 53-78 - - Malaysia 13
22 22 272 27 57 - - India 14
20-30 24-34 ? 24-30 36-57 - - Venezuela 15
21-26 27-28 284-302 21-29 46-62 - - Vietnam = =16
19-21 21-22 280-319* ? 53-58 = - - India 17
22-27 22-27 ? ? 50-79 - - India 11
20-23 24-27 232-297 22-28 45-55 - - India 14
20-21 22-23 278-293 22-24 53-60 - - Vietnam 16

'References: 1 — Carvalho (1956). 2 — Mulvey (1963). 3 — Cobb (1917). 4 — Williams (1958). 5 — Lordello (1970). 6 — Ahmad and Jairajpuri (2010). 7 — Carvalho (1951).
8 — Mulvey and Jensen (1967). 9 — Chaves and Geraert (1977). 10 — Mohandas and Prabhoo (1979). 11 — Jairajpuri and Khan (1982). 12 — Chaves (1990). 13 — Loof
(2006). 14 — Tahseen ef al. (2013). 15 — Perichi et al. (2021). 16 — Present paper. 17 — Jairajpuri (1971).

*Morphometrics of specimens collected from two or more locations.
* Values calculated from literal description and/or other morphometrics.

? = Either populations whose identity raises some doubt (see text) or morphometrics not available from the corresponding reference.

of small teeth with decreasing size from the base till the
level of dorsal tooth. Anterior end of pharynx embracing
the basal part of buccal cavity, gland nuclei obscure. Nerve
ring located at 93-101 um or ca one-third (34-36%) of the
total neck length. Pharyngo-intestinal junction non-tuber-
culate, 15 x 8-9 um. Genital system diovarian, with small
and equally developed branches occupying 6—7% of body
length: ovaries comparatively large, 50-56 um long, with
oocytes first arranged in several rows and then in only one
row; genital tract very short: oviduct 40-57 um long or
1.1-1.4 body diameters, consisting of a small distal part
and a well-developed pars dilatata, sphincter hardly per-
ceptible, uterus a simple tube 17—21 um or 0.4—0.5 body
diameters; vagina 11-12 um long, extending inwards
to less than one-third (29-30%) of body diameter, pars
proximalis 5.5—6.5 x 1.5—2 um, pars refringens with two
trapezoidal sclerotized pieces 2.5 x 2 um and a combined
width of 4.5—5.5 um, pars distalis 0.5—-1.5 um, vulva a
transverse slit. Rectum as long as anal body diameter long.
Tail conical with finely rounded tip, regularly curved ven-
trad, with distinct caudal glands leading to a visible am-
pulla with terminal spinneret 2.5 um long (Figs 3, 4).

Male. Not found.

Locality and habitat. Vietnam, Quang Ninh Prov-
ince, Bach Long Vi Island, where the nematodes were
collected in soil around the roots of a natural forest.

Voucher specimens. Permanent slides are stored at
the Department of Nematology, Institute of Ecology and
Biological Resources, VAST, Hanoi, Vietnam.

Remarks. As mentioned in the introductory section,
these specimens were originally described as 4. conoidus

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by Vu (2017). Nevertheless, their general morphology and
morphometrics are almost identical to those provided by
Jairajpuri (1971) for the type material of S. vagabundus,
with no appreciable difference. This species is very similar
to S. ibitiensis, but the study of Vietnamese specimens of
both species have revealed some relevant differences. Mor-
phologically, S. vagabundus females show a more devel-
oped pars dilatata oviductus and bear distinct caudal glands
that, most importantly, lead to appreciable terminal ampulla
and spinneret (vs ampulla and spinneret absent). Morpho-
metrically (Table 1), S. vagabundus shows a much shorter
buccal cavity (22—23 vs 27—28 um). Although these differ-
ences are based on the comparison of only a few specimens,
they seem significant enough to separate both species.

Jairajpuri’s (1971) original description and Vietnamese
specimens herein studied are regarded as the basic material
for the characterization of this species and for comparative
purposes. Other references should be taken with more cau-
tion due to some uncertainties. Jairajpuri and Khan (1982)
provided data of specimens with excessively large ranges
of some relevant morphometrics (for instance, L = 1.2—1.8
mm, buccal cavity 22—27 um), which might belong to
more than one species. Tahseen et al. (2013) studied two
Indian populations with smaller general size (body length
0.91—1.09 mm), and some inconsistences in their descrip-
tion (for instance, buccal cavity 1.4—1.6 times wider than
long according to literal description, but 24-27 x 11-13 um
after the morphometrics provided in their Table 1, and buc-
cal cavity similar-sized after their morphometrics (25—27
and 24—26 um long), but appreciably different according
to Figs 1A, 2A and their corresponding scales.

Zoosyst. Evol. 100 (1) 2024, 155-166

161

Figure 3. Sporonchulus vagabundus Jairajpuri, 1971 (Female). A. Entire; B, C. Anterior body region, lateral median view; D. Neck
region; E. Posterior genital branch; F. Anterior body region, lateral surface view; G. Caudal region; H. Pharyngo-intestinal junction;
I. Vagina. Scale bars: 200 um (A); 5 um (B, C, F, G, H); 50 um (D); 10 um (KE, G).

Evolutionary relationships of Sporonchulus
Cobb, 1917

The study of Vietnamese females of S. ibitiensis and
S. vagabundus confirms at least two remarkable mor-
phological features with evolutionary projection: the
absence of tubercles at their pharyngo-intestinal junction
and, most important, its buccal cavity, very similar to that
found in Mononchidae members. Thus, the buccal cav-
ity consists of more or less parallel, convergent at their
both ends, vertical (anterior) plates, and visibly oblique
horizontal (posterior) plates. Besides, the dorsal tooth,
located at the anterior third of buccal cavity, shows an

almost horizontal plane anterior margin, very slightly for-
ward directed. This design significantly differs from that
observed in Mylonchulidae, with the vertical and basal
plates more distinctly converging at their posterior ends
than at their anterior ends, the whole cavity becoming
V-shaped, and the dorsal tooth conspicuously forwards
directed, its anterior margin appearing visibly concave.
The mylonchulid pattern is herein interpreted as derived
(apomorphic) compared to the more primitive (plesiom-
orphic) mononchid (Mononchidae) pattern.
Unfortunately, the GenBank database does not include
any representative of Sporonchulus, therefore the first
sequences of both 18S and 28S rDNA are herein provid-

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162 Vu, T.T.T. et al.: The new insights into the evolutionary relationships of the genus Sporonchulus

Figure 4. Sporonchulus vagabundus Jairajpuri, 1971 (Female, LM). A. Entire; B—D. Anterior body region, lateral median
view; E. Caudal glands; F—H. Anterior body region, lateral submedian view; I. Vagina; J. Caudal region; K. Posterior genital
branch; L, N. Anterior body region, lateral surface view; M. Detail of caudal region tip; O. Pharyngo-intestinal junction. Scale bars:

200 um (A); 5 um (B+, L, N, O); 10 um (J, K, MD.

ed. The results of their analyses are presented in the trees
shown in Figs 5, 6, respectively. A BLAST search for
matches to the partial 18S rDNA sequence revealed 99%
similarity to Actus sequences, 97-98% to Miconchus sp.,
96.6% to lotonchulus, 96% to several Mylonchulus se-
quences, etc. 28S rDNA sequence is 98% similar to that of

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Actus, 94-95% to Coomansus, Parkellus and Prionchulus,
93-94% to Anatonchus, 86-88% to Mylonchulus, etc. Both
trees display a different main branching of Mononchina
representatives. 18S tree presents Mononchus sequences
(Mononchidae, Mononchinae) forming part of a totally
supported (100%) clade that is the sister group of the re-

Zoosyst. Evol. 100 (1) 2024, 155-166

97 |
93

100
97

9

98

100

96

93

99

99

100

90 |

| |
99

91.

100 |

— KJ6363

163

100, AB361445 Mylonchulus oceanicus
AB361443 Mylonchulus oceanicus
AY284751 Mylonchulus rotundicaudatus
r— AY284756 Mylonchulus sigmaturus
AB361437 Mylonchulus brachyuris
— AY284757 Mylonchulus sigmaturus

OP077090 Mylonchulus hawaliensis

92 ' AB361442 Mylonchulus hawaiiensis
JQ742964 Mylonchulus hawaiiensis
AF036596 Mylonchulus arenicolus

100 AFOQ36596 Mylonchulus arenicolus

AB361449 Mylonchulus mulveyi
100 AB361448 Mylonchulus mulveyi

-—— 0Q377123 Sporonchulus ibitiensis
— AB361035 Actus salvadoricus
-— MT799669 Coomansus parvus
MT799665 Parkellus hagiangensis
MT799667 Parkellus zschokkei
100; AB361452 Clarkus papillatus
' AJ966479 Clarkus sp. PDL-2005
AJ966500 Prionchulus muscorum
__ > AY284747 Prionchulus punctatus
100 AY284746 Prionchulus punctatus
-— AJ966474 Anatonchus tridentatus
— KJ636436 Miconchus sp. 1 JH-2014
98 MW218936 lotonchus lotilabiatus sp. nov.
100 ' MW218937 /otonchus /otilabiatus sp. nov.
AY593954 Mononchus tunbridgensis
AY284762 Mononchus truncatus
KJ636355 Mononchus truncatus
|__; KJ636382 Mononchus pulcher

83 Mononchus aquaticus

0.020

AY284744 Bathyodontus mirus

Figure 5. Bayesian Inference tree from the newly sequenced Sporonchulus ibitiensis (Carvalho, 1951) Andrassy, 1958 based on

sequences of the 18S rDNA region. Bayesian posterior probabi
substitutions per site.

maining members of the suborder, all of them components
of another, highly supported (98%) clade. Conversely, 28S
tree presents Mylonchulus sequences (Mylonchulidae, My-
lonchulinae) forming a maximally supported clade, which
is the sister group of the remaining taxa included in anoth-
er, maximally supported clade. It is remarkable that neither
of these two trees confirms the traditional (morphological)
division of Mononchina into two superfamilies, name-
ly Anatonchoidea and Mononchoidea, a system accepted
for several decades (Jairajpuri 1969; Jairajpuri and Khan
1982: Andrassy, 1976, 1993, 2009), which did not obtain
confirmation when the first molecular analyses, focused on
SSU rDNA (Holterman et al. 2006, 2008; Olia et al. 2008;
van Megen et al. 2009), were available. Regarding the po-
sition of Sporonchulus sequences in Mononchida tree, both
trees significantly differ in their topology. Thus, 18S tree
shows Sporonchulus sequence as part of a highly supported
(99%) clade also including Actus salvadoricus, this clade
being the sister group of a maximally supported (100%)
Mylonchulus clade, both clades grouped in a less support-
ed major (96%) clade. These results argued in favour of

lities (%) are given for each clade. Scale bar shows the number of

the monophyly of Sporonchulinae and of its inclusion in
the family Mylonchulidae. Conversely, in LSU tree the
Sporonchulus sequence is included in a weakly supported
(85%) large clade together with Mononchus sequences,
which Is the sister group of a maximally supported clade in-
cluding all the sequences belonging to Anatonchoidea gen-
era (Anatonchus, lotonchus, Miconchus, Parahadronchus).

As mentioned in the introductory section, the position
of Sporonchulus and Sporonchulinae was a matter of some
controversy. Several authors (Andrassy 1976, 1993, 2009;
Zullini and Peneva 2006) claimed they belonged to Monon-
chidae. This hypothesis is herein morphologically support-
ed. Conversely, molecular analyses do not agree about the
position of Sporonchulus sequences (see above), but 28S
tree provides better resolution in the main branching of
Mononchina, with S. ibitiensis sequence forming part of the
large clade which is the sister group of Mononchus clade,
thus supporting the results dertved from morphological
analysis. Although a further study should be conducted to
clarify the subject, the belonging of Sporonchulus and Spo-
ronchulinae to Mononchidae 1s herein tentatively accepted.

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164 Vu, T.T.T. et al.: The new insights into the evolutionary relationships of the genus Sporonchulus

100

100

0Q132796 Mylonchulus_index

100

ON927922_Mylonchulus_sigmaturus

MW227656_Prionchulus_jonkershoekensis

100

100

100

MG994945_Prionchulus_punctatus

100
100 MIW525198_Coomansus_batxatensis
100

M1T799673_Coomansus_parvus
100
100;-— MT799670_Parkellus_hagiangensis

160

100

100

0.06

100

0
—— MT/99671_Parkellus_tuyenquangensis

100
—— MT799672_Parkellus_zschokkei

bh AY593065_Anatonchus_tridentatus
98 OP877775_lotonchulus_longicaudatus
gg a 0Q170964_lotonchus_trichurus
100 nl OP997552_lotonchus_parabasidontus
100 92 0Q170962_Parahadronchus_divendentus
— Foo
— OP866150_Parahadronchus_siroii
a ON927927_Miconchus_elegans
100 ae AY593064_Mononchus_truncatus

100
AY593063_Mononchus_tunbridgensis
100

0Q377128 Sporonchulus ibitiensis

EF417153_Romanomermis_culicivorax

M2494738 Nematimermis_enoplivora

Figure 6. Bayesian Inference tree from the newly sequenced Sporonchulus ibitiensis (Carvalho, 1951) Andrassy, 1958 based on
sequences of the 28S rDNA region. Bayesian posterior probabilities (Y%) are given for each clade. Scale bar shows the number of

substitutions per site.

Taxonomy of Sporonchulus Cobb, 1917
Sporonchulus Cobb, 1917

= Sporonchuloides Mohandas & Prabhoo, 1982, syn. by
Andrassy (1993).

Historical outline. Cobb (1917) proposed the subgenus
Sporonchulus under the genus (Mononchus) to accommo-
date three new species, namely M. (S.) decurrens, M. (S.)
dentatus (type) and M. (S.) recessus, and characterized it
by the presence of irregularly arranged denticles opposed
to the dorsal tooth. Andrassy (1958) raised the rank of Spo-
ronchulus to generic level, regarded M. ibitiensis Carvalho,
1951 and M. coronatus Carvalho, 1956 as members of Spo-
ronchulus, and transferred M. (S.) decurrens to Granonchu-
lus, and M. (S.) recessus to Judonchulus. Since then, the
taxonomy of the genus has been a matter of some contro-
versy. Goodey (1963) follows Andrassy’s ideas. Mulvey
(1963) listed five species, recovering S. recessus and de-
scribing a new species, S. minutus. Jairaypuri (1971) added
a sixth species, S. vagabundus, and noted that the genus
consisted of two groups of species, but he did not propose
any nomenclatorial change. Jairaypuri and Khan (1982) in-
cluded only four species (coronatus, dentatus, ibitiensis and
vagabundus), a scheme accepted in subsequent contribu-
tions (Andrassy 1993, 2009; Ahmad and Jairajapuri 2010).

Mohandas and Prabhoo (1982) created the genus Spo-
ronchuloides to transfer two Sporonchulus species, namely

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ibitiensis (type) and coronatus, with “at least two (of their
denticles) arranged in longitudinal ribs”, meanwhile re-
tained under Sporonchulus three species: dentatus, re-
cessus and vagabundus. Moreover, Sporonchuloides was
classified under Mononchidae instead of under Mylonchu-
lidae. Nevertheless, Andrassy (1993) discussed the identity
of this genus and concluded that the denticles of S. ibitien-
sis were “not arranged along ribs”, and regarded Spronchu-
loides as identical and a junior synonym of Sporonchulus.
Diagnosis. Mononchidae. Sporonchulinae. Small- to
medium-sized nematodes, 0.95—-1.78 mm long. Cuticle
two-layered. Lip region almost continuous with the ad-
joining body, with fused lips. Buccal cavity with slightly
convergent vertical plates and visibly oblique transverse
plates, dorsal tooth situated at the anterior third of the
cavity, each vertical subventral plate bearing two irregu-
lar rows of small teeth, ca 24 in total. Pharyngo-intestinal
junction lacking tubercles. Female genital system diovar-
ian or mono-opistho-ovarian, with poorly differentiated
genital tract, distinct pars refringens vaginae and a trans-
verse vulva. Tail conical, regularly curved ventrad, with
variably developed caudal glands and terminal spinner-
et. Males very rare, with dorylaimid spicule and 11-12
shortly spaced ventromedian supplements without hiatus.
Separation from its relatives. Within Sporonchulinae,
and by having dorsal tooth located at the anterior third of
buccal cavity, Sporonchulus resembles the genera Granon-
chulus and Actus Baqri & Jairajpuri, 1974. It can be distin-
guished from Granonchulus in the absence (vs presence)

Zoosyst. Evol. 100 (1) 2024, 155-166

of a transverse row of denticles on the vertical subventral
plates at level of the dorsal tooth, and small teeth arranged
in four (two on each plate) irregular longitudinal rows
(vs teeth scattered, not distinctly arranged in longitudinal
rows) on the vertical subventral plates. From Actus in bear-
ing higher number of subventral teeth (ca 24 vs ca 10) ar-
ranged into four (vs two) irregular longitudinal rows, two
rows per subventral plate (vs only one row per plate).

Type species:

S. dentatus Cobb, 1917
= Mononchus (Sporonchulus) dentatus Cobb, 1917

Key to species

1 Female genital system mono-opistho-ovarian ..............0.08

165
Other species:

S. coronatus (Carvalho, 1956) Andrassy, 1958
= Mononchus coronatus Carvalho, 1956
= Sporonchuloides coronatus (Carvalho, 1956) Mo-
handas & Prabhoo, 1982

S. ibitiensis (Carvalho, 1951) Andrassy, 1958
= Mononchus ibitiensis Carvalho, 1951
= Sporonchuloides ibitiensis (Carvalho, 1951) Mohan-
das & Prabhoo, 1982

S. vagabundus Jairajpuri, 1971

- Pelriale Semitalt SVSTEHT Cl MOW el All m. 2 Sis x no8d ees sew FeNcee WA rae Sys Foley 9 Pe oe ars ae NN Ree een Sad Wee oles 208 Rel eee. Sed one 2a NN See D alk ead 2
2 Pars dilatata oviductus well-developed. Caudal glands conspicuous, with terminal aperture at tail end ....... S. vagabundus
~ Pars dilatata oviductus hardly appreciable. Caudal glands and their aperture INCONSPICUOUS .............0:ceeeeeeeeee ness eeee teens S|
3 LALSEMOUCK al deals O25 Sri WO eects: yall MAW, eels ORR coe Bs das) RA ME eDiets OOO UR OR ete et MO S. dentatus
- smallerbuccal cavity, up:to:sO: pm long; onlyexceptionally: MOKe-. .... 0. pcclveneseerilge ses ccgeereucenntensgledd eendalelgecetaureiues S. ibitiensis

Table 2 compiles the main morphometrics of known
populations of Sporonchulus species for comparative
purposes.

Acknowledgements

The Spanish author (RPS) deeply appreciates the invita-
tion, support and reception of the Department of Nem-
atology, Institute of Ecology and Biological Resources
(IBSR), Vietnamese Academy of Sciences and Technol-
ogy (VAST), Hanoi, Vietnam, to visit its lab and to par-
ticipate in the project “Developing the first-class research
team on the discovery of diversity and application poten-
tial of hymenopterans, myriapods and soil nematodes in
the limestone mountains of north-eastern Vietnam” grant
number NCXS01 .04/23-25.

The authors would like to thank Dr. Tran, Duc Luong
(Institute of Ecology and Biological Resources, VAST)
for supporting the value of SEM photographs.

The work is supported by the Vietnam Academy
of Science and Technology (VAST) under the project
NCXS01.04/23-25 “Developing the first-class research
team on the discovery of diversity and application poten-
tial of hymenopterans, myriapods and soil nematodes in
the limestone mountains of north-eastern Vietnam”.

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