CC BY
7Russian Journal of Nematology, 2023, 31 (2), 89 - 99
Morphological and molecular characterisation of
the Mexican cyst nematode, Globodera mexicana
Subbotin, Mundo-Ocampo & Baldwin, 2010
(Tylenchida: Heteroderidae)
1 12 Ignacio Cid del Prado Vera , Josue A.M. Ceron , Valeria Orlando ,
Rebecca Lawson2, Thomas Prior2 and Sergei A. Subbotin3
'Colegio de Postgraduados, 56230, Montecillo, Mexico 2Fera Science Ltd., Sand Hutton, YO41 1LZ, York, UK 3Plant Pest Diagnostic Center, California Department of Food and Agriculture, 95832-1448, Sacramento, CA, USA
e-mail: [email protected]
Accepted for publication 28 June 2023
Summary. The Mexican cyst nematode, Globodera mexicana, was reported from wild solanaceous plants collected from several locations in Mexico. The analysis of COI, cytb, ITS rRNA and hsp90 gene sequences suggested that G. pallida and G. mexicana are sister species. In this study, 15 new COI, three new cytb, six new hsp90, and two new ITS rRNA gene sequences were provided. Maximal intraspecific COI gene sequence diversity for G. mexicana was estimated as 9.9%. Morphological description of G. mexicana from type and another location was also provided. Considering our current knowledge on findings of G. mexicana, it has been suggested that G. mexicana originated and diversified in Mexico and the cysts of its ancestor have dispersed to Mexico by passing through the northern Andes and Central America. Future studies of the genus Globodera should focus on increased sampling in these regions of Central and South America from wild solanaceous plants to reconstruct a more complete picture of phylogeography and evolution of this genus.
Key words: biogeography, COI, cytb, Globodera, Mexico, phylogeny.
In 1963, during nematological surveys of wild potatoes and solanaceous plants in the Central Highlands of Mexico's Toluca Valley in the state of Mexico and Huamantla Valley in the state of Tlaxcala, many cysts closely resembling potato cyst nematodes were found from Solanum rostratum Dunal. Detailed biological and morphological studies made by Campos-Vela (1967) revealed this nematode represented an undescribed species. The data from this work, together with a description of a species named as Heterodera mexicana, was presented in Campos-Vela's Ph.D. thesis. Several years later, however, Golden and Ellington (1972) considered H. mexicana as a nomen nudum as the description of this nematode was presented in a Ph.D. thesis, which could not be considered as a valid publication for the purposes of species nomenclature. Considering that, the Mexican cyst nematode is biochemically and molecularly different from other Globodera, Subbotin et al. (2010) proposed to re-establish this species by accepting it as a valid species and naming it G. mexicana.
Experiments showed that G. mexicana was unable to develop on potato (Solanum tuberosum), whereas tomato (Solanum lycopersicum) was a common host for G. pallida and G. mexicana (Thiery et al., 1997). These two species were able to mate and form hybrids (Mugniery et al., 1992). Results of two-dimensional gel electrophoresis of total proteins (Bossis & Mugniery, 1993), PCR-ITS-RFLP (Thiery et al., 1997; Grenier et al., 2002), PCR-RAPD (Thiery et al., 1997), and satellite DNA sequences (Grenier et al., 2002) showed that the G. mexicana was clearly different from all other Globodera spp. and shared a high degree of genome similarity with G. pallida. Several genes responsible for parasitism and interaction with plants were characterised in G. mexicana and compared with those from G. pallida (Grenier et al., 2002; Blanchard et al., 2005; Sacco et al., 2009; Stare et al., 2011) and sequences of cytb (Picard et al., 2007), ITS rRNA (Picard et al., 2008; Subbotin et al., 2011), and 18S rRNA (Helder et al., 2014) genes were also published. Eight populations of
© Russian Society of Nematologists, 2023; doi: 10.24412/0869-6918-2023-2-89-99
G. mexicana, including the topotype population, were recently analysed using ITS rRNA, cytb and COI gene sequences (Subbotin et al., 2020).
In this study, we provide morphological descriptions of the topotype population of G. mexicana and analyse additional populations of this species. Updated phylogenetic networks for COI and cytb genes and a phylogenetic tree showing relationships within G. pallida and G. mexicana based on the analysis of hsp90 gene sequences are also provided.
MATERIAL AND METHODS
Nematode samples. In September 2021, a nematological survey was conducted across Central Mexico. More than 50 soil samples from solanaceous and other plants were collected from several locations. Adult and second-stage juvenile (J2) cyst nematodes were also collected from the type locality of G. mexicana in Tlaxcala State, Huamantla, Francisco Villa Tecoac. Cysts and J2 were extracted from soil samples using standard centrifugal-flotation and the Fenwick methods (Fenwick, 1940). Several dozen cysts were obtained from Stone's nematode collection presently kept at Fera Science Ltd., York, UK. They were also included in this study. These cysts were collected during a visit to Mexico in November 1975 (Table 1). For molecular analysis, several other G. mexicana populations characterised in a previous publication (Subbotin et al., 2020) were also used.
Morphological study. Males, females and J2 were killed using 8% formalin. The nematodes were then processed to glycerin using a modification of the Seinhorst (1959) method. Morphometrics of certain characters were calculated following schematic drawings made using a drawing tube mounted on an American Optical Compound Microscope (AO, USA). Light micrographs of cysts and J2 were taken with automatic Lumenera Infinity 2 camera (Japan) attached to a compound Olympus BX51 microscope (Japan) equipped with Nomarski differential interference contrast.
For scanning electron microscopy, nematodes were treated in phosphate buffer for 15 min and dehydrated in an alcohol series (10-100%) for 15 min at each concentration (Cid del Prado Vera et al., 2012). The specimens were critical point-dried and coated with gold-palladium before observation under a scanning electron microscope, Jeol JSM-6390 (Japan) at 10 kV.
DNA Extraction, PCR and sequencing. Cysts were soaked for 10-20 min in double distilled water (DDW). One cyst was placed in 20 ^l DDW on a glass slide, punctured by a needle under a dissecting microscope to release J2 and eggs, which were then cut using a stainless-steel dental needle under a stereomicroscope. DNA was extracted using a standard protocol with proteinase K (Subbotin et al., 2020). Fragments of nematodes in the water suspension were transferred into a 0.2 ml Eppendorf tube and 3 ^l proteinase K (600 ^g ml-1) (Promega) and 2 ^l 10X PCR buffer (Taq PCR Core Kit, Qiagen) were added to each tube. The tubes were incubated at 65°C (1 h) and 95°C (15 min) consecutively. After incubation, the tubes were centrifuged and kept at -20°C until use.
PCR and sequencing were performed as described by Subbotin et al. (2020). Several primer sets were used: the forward Het-coxiF (5'-TAG TTG ATC GTA ATT TTA ATG G-3') and the reverse Het-coxiR (5'-CCT AAA ACA TAA TGA AAA TGW GC-3') primers for amplification of the partial COI gene; the forward Het-cytbF2 (5'-CAR TAT TTR ATR TTT GAR GT-3') and reverse Het-cytbR3 (5'-ACH ARR AAR TTR ATY TCC TC-3') primers for amplification of the partial cytb gene; the forward TW81 (5'-GTT TCC GTA GGT GAA CCT GC-3') and the reverse AB28 (5'-ATA TGC TTA AGT TCA GCG GGT-3') for amplification of the ITS1-5.8S-ITS2 rRNA gene; and, the forward U831 (5'-AAY AAR ACM AAG CCN TYT GGAC-3') and the reverse L1110 (5'-TCR CAR TTV TCC ATG ATR AAV AC-3') for amplification of the hsp90 gene. The PCR products of several samples were cloned into the pGEM-T vector and transformed into JM109 High Efficiency Competent Cells (Promega, USA). Sequencing was performed by Genewiz (San Francisco (CA), USA). New sequences were submitted in GenBank under accession numbers indicated in Table 1.
Phylogenetic analysis. Alignments of the COI, cytb, ITS rRNA and hsp90 gene sequences were created using ClustalX 1.83 (Chenna et al., 2003) with default parameters. New sequences were aligned with corresponding published gene sequences (Subbotin et al., 2020). Several alignments were created: i) COI gene alignment containing sequences of G. mexicana; ii) cytb gene alignment containing sequences of G. mexicana; iii) ITS rRNA gene alignment containing sequences of G. mexicana and some sequences of other Globodera species parasitising solanaceous plants;
Table 1. Populations of Globodera mexicana used in this study.
Location GPS coordinates Plant-host Sample code Haplotypes GenBank accession number Source
ITS rRNA COI cytb hsp90
Mexico, State of Mexico, Juchitepec County 19.11159 -98.93599 Solanum pubigerum CD3563, M32 GmexCOI3/ GmexCb4 OQ318153 OQ318069-OQ318071 OQ320750 OQ332473 I. Cid del Prado Vera
Mexico, State of Mexico, Santiago Cuaula 19.57835 -98.64185 S. nigrescens CD3564, M49 GmexCOI5/ GmexCb5 OQ318154 OQ318074-OQ318076, OQ318079 OQ320748 OQ332475 I. Cid del Prado Vera
Mexico, State of Tlaxcala, Santiago Cuaula 19.57835 -98.64185 S. pubigerum CD3565, M50 GmexCOI5 - OQ318068, OQ318072, OQ318073, OQ318077, OQ318078 - - I. Cid del Prado Vera
Mexico Unknown Unknown CD3602 GmexCOI8/ GmexCb6 - OQ318080-OQ318082 OQ320749 - V. Orlando
Mexico, State of Mexico, Texcoco, El jardin de Tequexquinahuc 19.45532 -98.77861 S. nigrum CD2821 GmexCOI2 MN258870 MN095874, MN095875 - OQ332472, OQ332474 Subbotin et al. (2020)
Mexico, State of Mexico, Aniecameca, San Diego, Huehuecalco 19.09263 -98.74487 S. stoloniferum CD2809 GmexCOI5 - MN095867 - OQ332471 Subbotin et al. (2020)
Mexico, State of Mexico, San Miguel de la Victoria 20.08520 -99.62422 S. nigrum CD2814 GniexCOIl MN258868 MN095877 - OQ332476 Subbotin et al. (2020)
Mexico, Tlaxcala State, Huamantla, Francisco Villa Tecoac 19.38455 -97.92855 S. rostratum CD2862 GniexCOI5, GniexCOI7, GmexCOI6 MN258871 MN095865, MN095866, MN095872 - - Subbotin et al. (2020)
o
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o'
02 a
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o
rt 3.
o »
o
Q o"
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o
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a a a
Fig. 1. Cyst and females of Globodera mexicana from the type locality. Scale bar = 500 ^m.
and iv) hsp90 gene alignment containing sequences of G. mexicana and some G. pallida sequences. Alignments of ITS rRNA and hsp90 gene sequences were analysed with Bayesian inference (BI) using MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003) as described by Subbotin et al. (2020). The best fit models of DNA evolution were obtained using the program jModelTest 0.1.1 (Posada, 2008) with the Akaike Information Criterion. The alignments for COI and cytb gene sequences were used to construct phylogenetic network estimation using statistical parsimony (SP) as implemented in POPART software (http://popart.otago.ac.nz) (Bandelt et al., 1999). The haplotypes were identified based on the SP results. Haplotype numbers are given according to Subbotin et al. (2020).
RESULTS
After screening 50 soil samples from solanaceous and other plants in Mexico, cysts and females of G. mexicana were found from three samples: CD3563, CD3564 and CD3565 (Table 1).
Morphological characterisation of Globodera mexicana. Two populations of this species: one
from the type locality and another one from the State of Mexico, Amecameca, San Diego, Huehuecalco were used for morphological description. Table 2 provides measurements of G. mexicana of these two populations, populations given by Campos-Vela (1967) and Franco et al. (2000) and G. pallida for comparison. Some morphological and morphometric characters used for diagnostics of Globodera parasitising solanaceous plants are given in Table 3.
Females. Mature white female pear to spherical shape with short neck, less than 120 ^m long; in some females a transparent-yellow secretion located around the neck was observed. Lip region with two cephalic rings, the first annuli 4 ^m in width and the second 5 ^m; cuticle with fine annulation and circumfenestra area lace-like in pattern. Vulval slit 10 ^m long and circumfenestra 14 to 15 ^m diam. Distance of the border of circumfenestra to anus 18 to 60 ^m. Stylet with conus curved dorsally. Procorpus with distortion by the gland's secretion. Metacorpus spherical in shape, in general 25 ^m diam. Pharyngeal glands forming a lobe, overlapping intestine; excretory pore posterior to metacorpus.
Table 2. Morphometries of cysts, second-stage juveniles and males of Globodera mexicana and G. pallida. All measurements are in цm and in the form: range (mean ± s.d.).
Mexico, Tlaxcala State, Huamantla, Francisco Villa Tecoac, type locality, Solanum rostratum, This study Mexico, State of
Population Mexico, Amecameca, Mexico, Tlaxcala Mexico, Mexico City, Jaltomata procumbens, ( = G. bravoae), Franco et al. (2000)
Character San Diego Huehuecalco, Solanum staloniferum, This study State, Huamantla, Francisco Villa Tecoac, type locality, Campos-Vela (1967) G. pallida, Handoo et al (2012)
Female
n 15 12 33 50 -
L 35G-645 (514.8±79.1) 4GG-7GG (5б1.3±93.7) 540-980 (б95.5) 152-83б (534±2б.7) -
W 2GG-472.5 (352.2±78.3) 217.5-542.5 (394.7±98.5) 350.4-б70.0 (483.б) Зб8-б20 (487.5±17.7) -
L/W 1.1-2.G (1.5±0.3) 1.2-1.8 (1.5±0.2) - 0.4-1.7 (1.4±0.1) -
Stylet 22-25 (24.2±1.3) 23-2б (24.3±1.5) 23.2-28.8 (24.7) 21.б-28.0 (24.5±0.7) 27.4±1.1
Length of metacorpus 25-35 (29.8±3.5) 22-35 (29.9±3.9) - 24.8-52.4 (З4.б±1.9) -
Width of metacorpus 25-3G (28.5±1.9) 25-35 (29.1±3.3) - - -
Cyst
n 4 б 100 40 -
L 412-52G (450.5±47.7) 312-б00 (383.7±1G7) б71-999 (815) 459-93б (739±35.б) 420-748 (578)
W 322-41б (372±43.б) 310-480 (355±б3.5) 429-800 (б35) 29б-749 (579±40.1) 400-б85 (535)
L/W 1.1-1.3 (1.2±0.1) 1.0-1.3 (1.1±0.1) - 1.1-1.б (1.3±0.1) -
Fenestra diam 11.G-27 (2G.6±5.9) 15-33 (22.7±4.8) 15.2-28.5 (20.9) 22-35 (27.8±1.1) 17.5-45.0 (23.7)
Anus to nearest fenestral margin distance 35-6G (45.8±9.б) 30-70 (4б.5±11.4) 34.2-110.0 (58.б) 34-80 (47.5±4.б) 30-80 (51.8)
Number of ridges between vulva and anus 9-15 (12±2.8) 7-14 (1G.4±2.2) - 8-15 (10.7) 7-17 (12)
Granek ratio 1.8-3.2 (2.3±0.б) 0.8-2.9 (2.1±0.5) 1.7-5.3 (2.8) 1.3-2.7 (1.8) 1.2-3.б (2.2)
Second-stage juvenile
n 5 5 125 59 -
L 318-411 (Зб4±41.5) 420-550 (47G±G.G5) 333-587 (4б8.5) 442-553 (484.б±б.9) 380-533 (4б8)
a 18.7-24.2 (22.8±2.3) 20.8-30.5 (25.4±3.5) 18.0-2б.0 (23.б) 20.4-31.1 (25.5±0.б) -
b 4.7-б.8 (5.4б±0.9) 5.4-б.2 (5.7±0.4) 2.1-3.7 (2.7) 4.3-4.8 (4.4±0.15) -
b' 3.4-5.1 (4.2±0.7) 2.8-3.3 (3.0±0.3) - 2.3-3.1 (2.б±0.1) -
c 2.б-4.7 (3.9±0.8) 7.4-9.2 (8.2±0.9) 5.7-11.б (8.б) 7.5-10.2 (8.б±0.1) -
c' 2.б-4.7 (3.9±0.8) 4.2-5.5 (5.0±0.5) - 4.2-б.2 (4.9±0.1) -
Stylet 21-23 (22±0.8) 23-24 (23.б±0.б) 20.0-27.0 (23.3) 18.4-2б.8 (22.б±0.5) 22.5-25.0 (23.5)
Stylet knob width 3.G-4.G (3.8±0.5) 4.0 - 3.б-б.0 (4.3±0.1) -
Lip region width 8-1G (9.2±G.8) 9.0-10 (9.б±0.б) - 8.0-11.б (9.2) -
Lip region height 4-5 (4.2±0.5) 3.0-5.0 (4.2±0.8) - 3.2-5.б (4.3±0.1) -
DGO 3.G 4.0-7.0 (5.3±1.5) - 5.б-8.0 (б.8±0.2) -
Length of metacorpus 8-12 (9.5±1.7) 12-14 (13.3±1.2) - - -
Primordium to anterior end 174-193 (183.5±13.4) 245-250 (247.5±3.5) 140-200 (175.7) 23б-305 (270±5.б) -
W (maximum) - - - 1б-24 (19.2±0.б) -
Excretory pore - 95-105 (98.3±5.8) 88.0-110.0 (100.4) - -
Tail 34-52 (4б.2±7.б) 50-б3 (5б.8±4.9) 44.2-74.2 (54.0) 45-бб (5б.2±1.1) 40-57 (51.0)
Hyaline part of tail length 25-31 (28.б±2.5) 25-34 (3G.2±4.G) 19.8-28.8 (24.5) 24.8-41.2 (32.2±1.0) 20-31 (2б.2)
Male
n 1 8 33 40 -
L 96g 1020-1190 (11GG±5G) 800-1300 (1150) 819-118б (1017±2б.б) 1198±104
a 2б.7 31-39 (Зб.7±2.5) 28.5-45.1 (35.9) 22.9-41.3 (32.5±1.9) -
b - 8.1-9.5 (8.9±0.5) 4.5-7.9 (б.1) - -
b' - 5.7-7.0 (б.4±0.б) - - -
c 192 159.3-278 (233.б±41) 130-547 (21 б) 134.9-459.7 (2б8.б±б.2) -
c' G.3 0.2-0.б (G.3±0.1) - 0.2-0.5 (0.3± 0.01) -
Stylet - 24-27 (25.5±0.9) 22-29.5 (2б.7) 18.8-28.0 (24.2 ±0.7) 27.5±1.0
Lip region width 1G.G 10-12 (10.б±0.7) - 8.4-12.4 (11.3±0.3) -
Lip region height б.0 5.0-10.0 (б.3±1.8) - 4.4-8.4 (б.2±0.4) -
DGO - 3.0-5.0 (4.G±G.8) - 4.0-б.4 (4.7±0.2) 3.4±1.0
Length of metacorpus - 12-18 (14.б±1.9) - - -
Excretory pore 120 135-1б0 (145.4±8.8) - 9б.4-1б9.9 (141.4±5.0) -
W (maximum) Зб 24-32 (2б.8±3.0) - 24.4-44.0 (32.1±1.б) -
Testis - 45-б3 (58.б±б.5) 51.2-б8.3 (бб.4) 22.5-бб.4 (44.9±11.9) -
Tail 5.0 2.0-б.0 (4.3±1.9) 2.0-8.8 (5.8) 2.4-7.б (4.1±0.4) -
Spicule length 28 22-34 (30±4.1) 27.8-3б.3 (33.9) 24-42 (31.1 ±1.2) 3б.3±4.1
Fig. 2. Cysts of Globodera mexicana. A, B: Cysts; C-F: Vulval plates. Scale bars: A = 500 цш, E, F = 20 цш.
Cysts. Spherical in shape, full of embryonate eggs. Tan-brown colour with a lace-like pattern surface. Subcrystaline layer absent. Small tubercules present around the vulva. Vulva fenestra circumfenestrate, bullae absent. Anus as a small pore, located dorsally (Figs 1 & 2).
Second-stage juveniles. Cephalic region rounded, separated from the body by constriction and with four
annuli, knobs anteriorly rounded or flattened. Pharyngeal glands fill the body cavity, overlapping intestine. Primordium slightly posterior to the mid-body; tail conical with pointed terminus (Fig. 3).
Males. Body ventrally arcuate following heat relaxation, approximately 1.0 mm long. Lip region rounded offset by a slight constriction. Metacorpus poorly developed and pharyngeal glands lobe shape,
Fig. 3. Second-stage juveniles of Globodera mexicana. A-E: Anterior end; F-J: posterior end. Scale bars = 10 ^m.
not occupying the width of body. Body annuli evident along the body and lateral field with four incisures. Tail very short.
Molecular characterisation of Globodera mexicana. Samples from different sources were used for molecular study (Table 1). Cysts of G. mexicana (CD3563, CD3564 and CD3565) were from three soil samples, whereas cysts of G. mexicana (CD3602) were from a glass vial under the name 'Mexican sample 178' in Stone's nematode collection. Additionally, three G. mexicana samples (CD2809, CD2814, CD2821) were used for amplification of hsp90 gene. Sequences of these samples were compared with those already published.
Phylogenetic and sequence analysis with COI gene. A total of 31 sequences of G. mexicana were analysed. Fifteen new COI gene sequences for this
species were obtained in this study. The alignment length was 443 bp. The haplotype network is given in Figure 4A with eight haplotypes revealed. One new haplotype, GmexCOI8, was identified in cysts from Stone's nematode collection. Maximal intraspecific COI gene sequence diversity for G. mexicana was 9.9%.
Phylogenetic and sequence analysis with cytb gene. A total of six sequences of G. mexicana were analysed. Three new cytb gene sequences for this species were obtained in this study. The alignment length was 516 bp. The haplotype network revealed six haplotypes (Fig. 4B). Three new haplotypes were designated in the present study for this species. Maximal intraspecific cytb gene sequence diversity for G. mexicana was 3.5%.
Phylogenetic and sequence analysis with ITS rRNA gene. A total of 27 sequences including two
Fig. 4. Statistical parsimony networks showing the phylogenetic relationships between COI (A) and cytb (B) haplotypes of G. mexicana. Small black circles represent missing haplotypes. Pie chart sizes are proportional to the number of samples with a particular haplotype. Nucleotide changes between haplotypes are given for appropriate branches. New sequences are indicated by bold letters.
new sequences of G. mexicana were analysed. Phylogenetic relationships between G. mexicana and other Globodera parasitising solanaceous plants as inferred from analysis of the ITS rRNA gene sequence alignment are given in Figure 5A. Sequences of G. mexicana formed a distinct cluster. Maximal intraspecific ITS rRNA gene sequence diversity for G. mexicana was 0.6%.
Phylogenetic and sequence analysis with hsp90 gene. A total of 22 sequences including six new sequences of G. mexicana were analysed. Phylogenetic relationships between G. pallida and G. mexicana parasitising solanaceous plants as inferred from analysis of the hsp90 gene sequence alignment are given in Figure 5B. Globodera mexicana occupied basal positions at this tree. Maximal intraspecific hsp90 gene sequence diversity for G. mexicana was 0.7%.
DISCUSSION
The results obtained in this study extended known morphometric variations for all stages of G.
mexicana. Comparative analysis of morphometrics of G. mexicana and G. pallida does not allow clearly differentiating these species from each other using morphological and morphometrical characters. Several molecular markers, sequences of ITS rRNA, hsp90, COI and cytb genes can distinguish these species.
The analysis of COI, cytb, ITS rRNA and hsp90 gene sequences suggested that G. pallida and G. mexicana are sister species with a divergence date of 1.6 million years ago (Subbotin et al., 2020). It has been proposed that G. mexicana originated and diversified in Mexico. After adding a new sample from the Stone's nematode collection from unknown Mexican location in the analysis, maximal intraspecific COI and cytb gene sequence diversities for G. mexicana were estimated as 9.9% and 3.5%, respectively, compared with 4.7% and 0.8% obtained in a previous study (Subbotin et al., 2020). The present study confirmed that the genetic diversity of G. mexicana is associated with zones of local topographical complexity. Mexico is crossed by large mountain systems (Sierra Madre Oriental,
Sierra Madre Occidental, Sierra Madre del Sur, Sierra de Chiapas, and the Trans-Mexican Volcanic Belt) corresponding to different geological provinces that differ vastly in age (Sosa et al., 2018). These mountains are hotspots of biodiversity and endemism as a result of local and regional
extinction, long-distance colonisation, and local recruitment (Subbotin et al., 2020).
Considering our current knowledge on findings of G. mexicana, we can suggest that the cysts of its ancestor have dispersed to Mexico by passing through the northern Andes and Central America.
Table 3. Some morphological and morphometric characters used for diagnostics of five Globodera parasitising solanaceous plants (measurements are given in ^m) after Subbotin et al. (2010), Handoo et al. (2012) and others.
Stage Cysts Second-stage juveniles
Character Species Number of cuticular ridges between fenestra and anus Granek's ratio Body length Stylet Hyaline part of tail Tail
G. mexicana 7-14 0.8-3.2 318-587 18-27 20-41 34-74
G. pallida 7-26 1.0-8.5 380-533 20-26 11-28 31-59
G. rostochiensis 12-31 1.3-9.5 425-505 19-24 20-27 44-51
G. ellingtonae 8-25 0.9-5.9 365-533 19-24 18-33 39-56
G. tabacum 5-15 1.0-4.2 410-527 19-28 21-28 50-56
A
Globodera mexicana (MN258873) Globodera mexicana (OQ318154, CD3564c) Globodera mexicana (OQ318153, CD3563c)
Globodera mexicana (EU006708) Globodera mexicana (EU006707) Globodera mexicana (MN116522) Globodera mexicana (EU006709) Globodera mexicana (HQ260405) Globodera mexicana (MN258872) Globodera mexicana (MN258869) Globodera mexicana (MN258870) Globodera mexicana (MN258868) Globodera mexicana (MN258871) Globodera mexicana (MN258874) 100|— Globodera tabacum (HQ260398) loop— Globodera rostochiensis (GQ294521) I— Globodera ellmgtonae (JF739888) Globodera pallida (HQ670264) Globodera pallida (MN475965) Globodera pallida (HQ670271) Globodera pallida (KY513117) Globodera pallida (HQ670269) Globodera pallida (GU084806) Globodera pallida (DQ847111) 90 L Globodera pallida (HQ670245)
Atalodera crassicrustata (HQ260425) Rhizonemella sequoiae (HQ260424)
_|— Punctodera stonei
В
r Globodera pallida (MK105555) - Globodera pallida (GQ401351) Globodera pallida (MK105556) Globodera pallida (JQ316193) Globodera pallida (MK105553) Globodera pallida (MK105557) <- Globodera pallida (MK105554) Globodera pallida (MK105558) Globodera pallida (HQ171927) 00 L Globodera pallida (GQ401350) r- Globodera pallida (MK105551) l Globodera pallida (MK105552) - Globodera pallida (KF834985) Globodera mexicana (OQ332475, CD3564c) L Globodera mexicana (OQ332476, CD2814, cl1) Globodera mexicana (OQ332471, CD2809, cl1) Globodera mexicana (OQ332474, CD2821 b, cl2) Globodera mexicana (OQ332473, CD3563c) Globodera mexicana (OQ332472, CD2821a, cl2) (MT661447) Punctodera punctata (MN182654) Punctodera matadorensis (MK580834)
Fig. 5. Phylogenetic relationships of Globodera mexicana with some Globodera species parasitising solanaceous plants: Bayesian 50% majority rule consensus trees from two runs as inferred from analysis of the ITS rRNA (A) and the hsp90 (B) gene sequence alignments under the GTR + I + G model. Posterior probabilities more than 70% are given for appropriate clades. New sequences are indicated by bold letters.
Future studies of the genus Globodera should focus on increased sampling in these regions of America from wild solanaceous plants to reconstruct more complete picture of phylogeography and evolution of this genus.
ACKNOWLEDGEMENTS
Authors thank J. Burbridge for technical assistance. This study was sponsored from the USDA APHIS Farm Bill grant AP20PPQS&T00C129 (agreement no. 20-0268-000-FR).
REFERENCES
Bandelt, H., Forster, P. & Röhl, A. 1999. Median-joining networks for inferring intraspecific phylogenies. Molecular Biology and Evolution 16: 37-48. DOI: 10.1093/oxfordjournals.molbev.a026036 Blanchard, A., Esquibet, M., Fouville, D. & Grenier, E. 2005. Ranbmp homologue genes characterized in the cyst nematodes Globodera pallida and Globodera 'mexicana'. Physiological and Molecular Plant Pathology 67: 15-22. DOI: 10.1016/j.pmpp.2005.09.001 Bossis, M. & Mugniery, D. 1993. Specific status of six Globodera parasites of solanaceous plants studied by means of two-dimensional gel electrophoresis with a comparison of gel patterns by a computed system. Fundamental and AppliedNematology 16: 47-56. Campos-Vela, A. 1967. Taxonomy, life cycle and host range of Heterodera mexicana n. sp. (Nematoda: Heteroderidae). Ph.D. Thesis, University of Wisconsin, USA, 70 pp. Chenna, R., Sugawara, H., Koike, T., Lopez, R. Gibson, T.J., Higgins, D.G. & Thompson, J.D. 2003. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Research 31: 3497-3500. DOI: 10.1093/nar/gkg5 00 Cid del Prado Vera, I. & Subbotin, S.A. 2012. Belonolaimus maluceroi sp. n. (Tylenchida: Belonolaimidae) from a tropical forest in Mexico and key to the species of Belonolaimus. Nematropica 42: 201-210.
Fenwick, D.W. 1940. Methods for the recovery and counting of cysts of Heterodera schachtii from soil. Journal of Helminthology 18: 155-172. Franco, N.F., I. Cid del Prado Vera, I. & Lamothe-Argumedo, R. 2000. Globodera bravoae sp. n. (Tylenchida: Heteroderidae) from Mexico. International Journal of Nematology 10: 169-176. Golden, A. M. & Ellington, D.M.S. 1972. Redescription of Heterodera rostochiensis (Nematoda: Heteroderidae) with a key and notes on closely related species. Proceedings of the
Helminthological Society of Washington 39: 64-77. Grenier, E., Blok, V.C., Jones, J.T., Fouville, D. & Mugniery, D. 2002. Identification of gene expression differences between Globodera pallida and G. mexicana by suppressive subtractive hybridization. Molecular Plant Pathology 3: 217-226. DOI: 10.1046/j.1364-3703.2002.00111.x Handoo, Z.A, Carta, L.K. Skantar, A.M. & Chitwood, D.J. 2012. Description of Globodera ellingtonae n. sp. (Nematoda: Heteroderidae) from Oregon. Journal of Nematology 44: 40-57. Helder, J., Mooijman, P J W., Elsen, S.J.J., van den Megen, H.H.B., Vervoort, M.T.W., Quist, C.W., Bert, W., Karegar, A., Karssen, G. & Decreamer, W. 2014. Biological and systematic implications of phylogenetic analysis of ~ 2,800 full length small subunit ribosomal DNA sequences. Proceedings of the 6th International Congress of Nematology, Cape Town, South Africa: 26. Mugniery, D., Bossis, m., & Pierre, J.-S. 1992. Hybridations entre Globodera rostochiensis (Wollenweber), G. pallida (Stone), G. virginiae (Miller and Gray), G. solanacearum (Miller and Gray) et G. 'mexicana' (Campos-Vela). Description et devenir des hybrides. Fundamental and Applied Nematology 15: 375-382. Picard, D., Sempere, T. & Plantard, O. 2007. A northward colonisation of the Andes by the potato cyst nematode during geological times suggests multiple host-shifts from wild to cultivated potatoes. Molecular Phylogenetics and Evolution 42: 308-316. DOI: 10.1016/j.ympev.2006.06.018 Picard, D., Sempere, T. & Plantard, O. 2008. Direction and timing of uplift propagation in the Peruvian Andes deduced from molecular phylogenetics of highland biotaxa. Earth and Planetary Science Letters 271: 326-336. DOI: 10.1016/j.epsl.2008.04.024 Posada, D. 2008. jModelTest: phylogenetic model averaging. Molecular Biology and Evolution 25: 1253-1256. DOI: 10.1093/molbev/msn083 Ronquist, F. & Huelsenbeck, J. 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572-1574. DOI: 10.1093/ bioinformatics/btg180 Sacco, M.A., Koropacka, K., Grenier, E., Jaubert, M.J., Blanchard, A., Goverse, A., Smant, G. & Moffett, P. 2009. The cyst nematode SPRYSEC protein RBP-1 elicits Gpa2- and RanGAP2-dependent plant cell death. PLOS Pathogens 5(8): E1000564. DOI: 10.1371/journal.ppat. 1000564 Seinhorst, J.W. 1959. A rapid method for the transfer of nematodes from fixative to anhydrous glycerin. Nematologica 4: 67-69. DOI: 10.1163/ 187529259X00381
Sosa, V., Arturo De-Nova, J. & VAsquez-Cruz, M. 2018. Evolutionary history of the flora of Mexico: Dry forests cradles and museums of endemism. Journal of Systematics and Evolution 56: 523-536. DOI: 10.1111/jse.12416 Stare, B.G, Fouville, D., Sirca, S., Gallot, A., Urek, G. & Grenier, E. 2011. Molecular variability and evolution of the pectate lyase (pel-2) parasitism gene in cyst nematodes parasitizing different solanaceous plants. Journal of Molecular Evolution 72: 169-181. DOI: 10.1007/s00239-010-9413-4 Subbotin, S.A., Mundo-Ocampo, M. & Baldwin, J.G. 2010. Systematics of cyst nematodes (Nematoda: Heteroderinae). In: Nematology Monographs and Perspectives, Volume 8A (D.J. Hunt & R.N. Perry Eds). 512 pp. Leiden, The Netherlands, Brill. SUBBOTIN, S.A., CID DEL PRADO, I., MUNDO-OCAMPO, M. & Baldwin, J.G. 2011. Identification, phylogeny and
phylogeography of circumfenestrate cyst nematodes (Nematoda: Heteroderidae) as inferred from analysis of ITS-rDNA. Nematology 13: 805-824. DOI: 10.1163/138855410X552661 Subbotin, S.A., Franco, J., Knoetze, R., Roubtsova, T.V., Bostock, R.M. & Cid DEL Prado Vera, I. 2020. DNA barcoding, phylogeny and phylogeography of the cyst nematode species from the genus Globodera (Tylenchida: Heteroderidae). Nematology 22: 269-297. DOI: 10.1163/15685411-00003305 Thiéry, M., Mugniéry, D., Bossis, M. & Sosa-Moss, C. 1997. Résultats de croisements entre Globodera pallida Stone et G. 'mexicana ' Campos-Vela: héritabilité du développement sur pomme de terre etnotion d'espèce. Fundamentals of Applied Nematology 20: 551-556. URL: http:/popart.otago.ac.nz (accessed: December 5, 2022).
I. Cid del Prado Vera, J.A.M. Ceron, V. Orlando, R. Lawson, T. Prior and S.A. Subbotin.
Морфологическая и молекулярная характеристика мексиканской цистообразующей нематоды, Globodera mexicana Subbotin, Mundo-Ocampo & Baldwin, 2010 (Tylenchida: Heteroderidae). Резюме. Мексиканская цистообразующая нематода, Globodera mexicana, была обнаружена на диких пасленовых растениях, собранных в нескольких местах Мексики. Анализ последовательностей генов COI, cytb, ITS рРНК и hsp90 позволил предположить, что Globodera pallida и G. mexicana являются сестринскими видами. В этом исследовании было предоставлено 15 новых COI, три новых cytb, шесть новых hsp90 и две новые ITS рРНК последовательности генов. Максимальное внутривидовое различие в последовательностях гена COI для G. mexicana оценивается в 9,9%. Дается морфологическое описание G. mexicana из типового места обитания и других районов. Принимая во внимание наши знания о находках G. mexicana, было высказано предположение, что этот вид возник и диверсифицировался в Мексике, а цисты его предка проникли в Мексику, миновав северные Анды и Центральную Америку. Будущие исследования по изучению биоразнообразия рода Globodera должны быть сосредоточены на увеличении количества сборов в этих регионах Центральной и Южной Америки с диких пасленовых растений. Это позволит воссоздать более полную картину филогеографии и эволюции этого рода.
