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Karyotype and genome size
analyses for two spiders of the
lycosidae family
Yuxuan Zhang
1
,
2
, Mengying Zhang
3
, Liang Leng
1
,
2
,YaWu
1
,
2
,
Hanting Yang
1
,
2
, Liangting Wang
1
,
2
, Baimei Liu
1
,
2
, Shuai Yang
2
,
Zizhong Yang
3
*, Shilin Chen
1
,
2
* and Chi Song
1
,
2
*
1
School of Pharmacy, Chengdu university of Traditional Chinese Medicine, Chengdu, China,
2
Institute of
Herbgenomics, Chengdu university of Traditional Chinese Medicine, Chengdu, China,
3
Yunnan
Provincial Key Laboratory of Entomological Biopharmaceutical R&D, College of Pharmacy, Dali
University, Dali, China
Background: Karyotype and genome size are critical genetic characteristics with
significant value for cytogenetics, taxonomy, phylogenetics, evolution, and
molecular biology. The Lycosidae family, known for its diverse spiders with
varying ecological habits and behavioral traits, has seen limited exploration of
its karyotype and genome size.
Methods: We utilized an improved tissue drop technique to prepare
chromosome slides and compare the features of male and female
karyotypes for two wolf spiders with different habits of Lycosidae.
Furthermore, we predicted their genome sizes using flow cytometry (FCM)
and K-mer analysis.
Results: The karyotypes of female and male Hippasa lycosina were 2n\=
26 = 14 m + 12 sm and 2n_= 24 = 10 m + 14 sm, respectively, and were
composed of metacentric (m) and submetacentric (sm) chromosomes. In
contrast, the karyotypes of Lycosa grahami consisted of telocentric (t) and
subtelocentric (st) chromosomes (2n\= 20 = 20th and 2n_= 18 = 12th + 6t, for
females and males). The sex chromosomes were both X
1
X
2
O. The estimated
sizes of the H. lycosina and L. grahami genomes were 1966.54–2099.89 Mb
and 3692.81–4012.56 Mb, respectively. Flow cytometry yielded slightly
smaller estimates for genome size compared to k-mer analysis. K-mer
analysis revealed a genome heterozygosity of 0.42% for H. lycosina and
0.80% for L. grahami, along with duplication ratios of 21.39% and 54.91%,
respectively.
Conclusion: This study describes the first analysis of the genome sizes and
karyotypes of two spiders from the Lycosidae that exhibit differential habits and
provides essential data for future phylogenetic, cytogenetic, and genomic
studies.
KEYWORDS
Lycosidae, tissue drop technique, Hippasa lycosina, Lycosa grahami, karyotype,
genome size
OPEN ACCESS
EDITED BY
Kornsorn Srikulnath,
Kasetsart University, Thailand
REVIEWED BY
Xia Liu,
Chongqing University of Arts and Sciences,
China
Cheng-Min Shi,
Hebei Agricultural University, China
*CORRESPONDENCE
Zizhong Yang,
yangzzh69@163.com
Shilin Chen,
slchen@cdutcm.edu.cn
Chi Song,
songchi@cdutcm.edu.cn
RECEIVED 12 December 2024
ACCEPTED 10 March 2025
PUBLISHED 25 March 2025
CITATION
Zhang Y, Zhang M, Leng L, Wu Y, Yang H,
Wang L, Liu B, Yang S, Yang Z, Chen S and
Song C (2025) Karyotype and genome size
analyses for two spiders of the lycosidae family.
Front. Genet. 16:1544087.
doi: 10.3389/fgene.2025.1544087
COPYRIGHT
© 2025 Zhang, Zhang, Leng, Wu, Yang, Wang,
Liu, Yang, Yang, Chen and Song. This is an open-
access article distributed under the terms of the
Creative Commons Attribution License (CC BY).
The use, distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in this
journal is cited, in accordance with accepted
academic practice. No use, distribution or
reproduction is permitted which does not
comply with these terms.
Frontiers in Genetics frontiersin.org01
TYPE Original Research
PUBLISHED 25 March 2025
DOI 10.3389/fgene.2025.1544087
1 Introduction
The family Lycosidae is one of the largest within the order
Araneae, with a global distribution, is known to comprise
2,474 species across 134 genera globally (https://wsc.nmbe.ch/
accessed on 12 September 2024). Wolf spiders, as predatory
species, hold significant research value in ecosystems, the
economy, and scientific research. In the Arctic tundra ecosystem,
they impact soil ecosystem functions through predation on other
insects and small invertebrates, playing a crucial role in maintaining
ecological balance (Koltz et al., 2018). The venom of Lycosidae
spiders contains various bioactive substances with antitumor,
antihypertensive, and antimicrobial properties, providing a rich
resource for the development of new drugs (Ma et al., 2018;Reis
et al., 2021;Tan et al., 2018). Furthermore, the chromosome number
and karyotype characteristics of Lycosidae species exhibit significant
diversity during evolution, which is of great importance for
understanding genetic diversity and evolution (Araujo et al.,
2015;Cavenagh et al., 2022).
Numerous spiders have evolved web-building skills to facilitate prey
capture, such as Hippasa lycosina (Figure 1A). In contrast, some spiders
remain active hunters, relying on speed and fast-acting venom to
subdue their prey, like Lycosa grahami (Figures 1B,C). These two
distinct predatory strategies enable them to occupy different
ecological niches, which may be related to species biodiversity (Koua
et al., 2020). Differences in chromosome numbers and genomic
characteristics are likely one of the important causes of biodiversity.
In spiders, chromosome numbers range from 2n = 24 in Trichonephila
clavata (_)(Suzuki, 1954)to2n=94inHeptathela kimurai (_)(Datta
and Chatterjee, 1988),andgenomesizesrangefrom0.82Gbin
Oedothorax gibbosus (Hendrickx et al., 2022)to6.79Gbin
Macrothele yani (You et al., 2024).Thesedifferencesmaybeclosely
related to the evolutionary adaptability and ecological diversity of
spiders (Ávila Herrera et al., 2021;Miles et al., 2024;Stávale et al., 2011).
The genome size and karyotype are pivotal cytogenetic
characteristics that have been extensively utilized in taxonomic,
phylogenetic, and evolutionary research (František et al., 2020;José
Paulo da Costa Pinto et al., 2020;Král et al., 2019;Wayne et al.,
2020). Chromosome karyotypes play a significant role in studying
species systematics, relationships, origins, evolution, and
classification (De Resende, 2017). However, previous karyotype
studies in the Lycosidae have primarily focused on chromosome
counts and sex chromosome behavior in males, with few
comparative analyses between sexes (Araujo et al., 2015;
Cavenagh et al., 2022;Chemisquy et al., 2008). To date, only
25 genera and 137 species within the family of Lycosidae have
been investigated at the cytogenetic level (https://
arthropodacytogenetics.bio.br/spiderdatabase/accessed on
12 September 2024) (Cavenagh et al., 2022;Chemisquy et al.,
2008)(Supplementary Table S1). Diploid chromosome numbers
in these species range from 18 to 30, with significant intrageneric
variation. For example, there are 22–28 chromosomes in males of
the Hippasa genera and 18 to 30 chromosomes in males from the
Lycosa genera. Notably, the same species can exhibit multiple
FIGURE 1
The habitats of the two spiders. (A) H. lycosina (large female and small male) are commonly found in rocky or soil crevices on slopes and spin large,
irregular funnel webs with non-sticky, polygonal meshes. (B) An example of a female L. grahami.(C) An example of a male L. graham i; this wolf spider does
not spin webs and frequents concealed areas such as rock crevices and soil cracks. (D) The feeding conditions provided in our laboratory. Note: The
image was provided by Professor Zizhong from Dali University.
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Zhang et al. 10.3389/fgene.2025.1544087
chromosome numbers, thus indicating potential aneuploidy, as
reported for H. deserticola (2n_= 26/28) and L. nigrotibialis
(2n_= 24/28) (Mittal, 1960;Mittal, 1963;Parida et al., 1986).
Genome size (C-value) is a key measure of genetic information
that is crucial for understanding genetic complexity and
evolutionary history and is closely associated with ecological
adaptation and evolutionary rates (Adachi et al., 2017;Olmo
et al., 2002;Zuo et al., 2023).
Currently, the determination of genome sizes in plants and
animals primarily relies on flow cytometry and genome survey
sequencing. Flow cytometry stands as the gold-standard
technique for genome-sizing in multicellular life forms (Wang
et al., 2015). The accuracy of this technique depends on the
availability of a species with a well-characterized genome size as
an internal reference, ideally one with a genome size close to and
stable in the target species (Dai et al., 2022). In the absence of ideal
plant or animal standard references, existing data are largely based
on subjectively selected standard species (Dolezel and Greilhuber,
2010). Despite the differences in cellular attributes between plants
and animals, plants can effectively serve as internal references for
animal genome size determination through meticulous
experimental design and parameter optimization. For example,
Hawlitschek et al. (2023) fine-tuned the nuclei lysis buffer
composition and successfully determined the genome size of
orthopteran insects using Pisum sativum (Fabaceae) as the
internal standard. Similarly, Král et al. (2019) employed Vicia
faba (Fabaceae) as the internal standard to estimate the genome
sizes of various spiders. With the advancement of sequencing
technology, genome survey sequencing can efficiently predict
genome size, heterozygosity, and the proportion of repetitive
sequences through k-mer analysis (Gao et al., 2022).
Consequently, to improve genome-sizing accuracy, flow
cytometry and genome survey sequencing are often combined for
cross-validation before genome sequencing, enabling appropriate
sequencing strategies (Gregory, 2005;Leng et al., 2024;Yang
et al., 2024).
Previous research of the family of Lycosidae predominantly
focused on taxonomy (Sankaran et al., 2017), biology (Naseem
and Tahir, 2018), ecology (Persons and Rypstra, 2000;Xiao et al.,
2015), and venom analysis (Abdel-Salam et al., 2021;Liu et al., 2021;
Myshkin et al., 2019) and did not consider karyotype or genome size.
Furthermore, previous research tended to focus more on male
chromosomes, with no comparative analyses reported between
sexes (Araujo et al., 2015;Cavenagh et al., 2022;Chemisquy
et al., 2008;Kumbıçak et al., 2009). In the present study, we
utilized flow cytometry and survey sequencing to determine the
genome sizes of two spiders from the same family but with different
predation strategies: H. lycosina and L. grahami and determine key
indices, including genome size, heterozygosity, and repetitive
sequences. In addition, we utilized the dropping technique for
karyotype investigation. In undertaking this research, we aimed
to establish a foundation for future cytogenetic and genomic studies
in the family of Lycosidae.
2 Materials and methods
2.1 Specimen acquisitions
Adult male and female spiders were collected from the field and
identified by Professor Zizhong (Table 1). The spiders were fed
Tenebrio molitor regularly on a weekly basis and water was provided
as needed to sustain their survival and maintain environmental
humidity (Figure 1D). Voucher specimens were deposited at the
Yunnan Provincial Key Laboratory of Entomological
Biopharmaceutical R&D, Dali University, Dali. Solanum
lycopersicum cv. Heinz 1706 was procured from Golden Future
FCM Biotechnology Inc., and young leaves, approximately two
months-of-age, were selected as an internal reference for flow
cytometry. To eliminate potential discrepancies arising from
variations in the number of sex chromosomes, female subjects
were selected for the estimation of genome size (Král et al., 2019).
2.2 Chromosome preparation
First, we performed a pre-experiment to optimize sampling,
colchicine pretreatment, hypotonic time, and staining duration to
generate metaphase plates from male H. lycosina, as described
previously (Ávila Herrera et al., 2021). In brief, we acquired
blood, legs, silk glands, gonads, and entire bodies of adult
spiders. Samples were then pretreated with 0.01%–0.1%
colchicine (prepared in RPMI 1640 complete medium) for 2 h at
room temperature (RT). Samples were then centrifuged at 2000 rpm
(5 min); then, the supernatants were removed, and the tissues were
immersed in hypotonic solution (0.075 mol/L KCl) for 0.5–2hat
RT. Following re-centrifugation, the tissues were placed in freshly
prepared and pre-cooled Carnoy’s solution (methanol: acetic acid (3:
1) 4°C for 30 min; this procedure was repeated on two further
occasions Subsequently, the tissues were transferred into 50% glacial
acetic acid and dissociated for 5 min. To generate chromosome
preparations, we employed the “dropping method”(Schmidt et al.,
2023), a simple and efficient protocol, where a cell nuclear
suspension is prepared and then dropped from a certain height
onto a slide, causing the nuclei to burst and the chromosomes to
spread. Finally, the slides were stained with 10% Giemsa (pH 6.8) for
10–30 min, rinsed three times with distilled water, dried naturally,
and finally sealed with neutral resin. For the analysis of meiosis in
TABLE 1 Information relating to the collection of spider specimens.
Species Number Collection locality Longitude Latitude Altitude (m)
Hippasa lycosina Pocock,1990 30\,50_Yangbi Yi Autonomous County, Yunnan Province, China 99°56′42″E25
°39′03″N 1718.1
Lycosa grahami Fox,1935 22\,20_Yuanma Town, Yuanmou X County, Yunnan Province, China 101°52′59″E25
°44′03″N 1140.0
Trichonephila clavata L. Koch, 1878 10\Wanqiao Town, Dali City, Yunnan Province, China 100°08′10″E25
°47′32″N 1937.7
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males, the testes were dissected and preserved in Carnoy’s solution
(methanol: acetic acid (3:1)) at 4°C. Chromosome smears were then
prepared and stained with 10% Giemsa. Finally, observations and
photographs were taken using an Olympus CX43 microscope
(Olympus, Nagano, Japan) at ×100 magnification.
2.3 Karyotype analysis
To evaluate chromosomal morphology and construct karyotypes,
30 mitotic metaphases were acquired from the slides generated from
each species. Five cells with clear morphology and well-dispersed
mitotic metaphases were selected, and karyotype analysis software
from Zeiss, Germany (Spot RT KE) was used to pair chromosomes
and measure their lengths (short arm length and long arm length). Data
were processed using Excel 2020 (Microsoft, Redmond, WA,
United States) and mean values from 5 cells were used as
parameters for karyotype analysis. Chromosome morphology was
described and analyzed based on chromosome arm lengths and
centromere positions, as described previously (Levan et al., 1964).
The karyotype classification refers to Stebbins (Stebbins, 1971). The
calculation formulas are as follows: Relative length (RL) = short arm
length + short arm length), Arm ratio (AR) = (long arm length/short
arm length), Chromosome length ratio (L/S) = longest chromosome
length/shortest chromosome length, Karyotype asymmetry coefficient
(As.K%) = (total length of long arms of chromosomes/total length of all
chromosomes) × 100%.
2.4 Flow cytometry and the estimation of
genome size
S. lycopersicum (2C = 0.85 pg (Sato et al., 2012)), a model species of
the Solanaceae, was used as an internal reference. In order to ensure the
reliability of our results, we used Trichonephila clavata (Araneidae) as a
positive control; the genome for this species has already been reported
(assembly size of 2.63 Gb and k-mer analysis of 2.72 Gb (Hu et al.,
2023)). These experiments were performed with CyStain PI Absolute P
(Seidl et al., 2022), spiders were sampled with legs, and S. lycopersicum
was sampled with fresh leaves, all other procedures were identical.
Samples were placed in 500 μL of nuclei extraction buffer and chopped
with a sharp blade, after 60 s, the samples were filtered through a 50 μm
filter, followed by incubation with 2000 μL of staining buffer containing
RNasefor15minindark.
Stained samples were then analyzed with a CyFlow Cube6 flow
cytometer (Sysmex Partec, Muenster, Germany) equipped with a
488 nm excitation light source. The fluorescence signals from at least
10,000 nuclei were acquired from each sample, and the coefficient of
variation (CV) was controlled to within 5% to optimize reliability
(Tomaszewska et al., 2021). Data were analyzed by FCSExpress
(v5.0) software. Three biological replicate measurements were
performed for each species to ensure the reliability of our results.
Sample DNA content = (mean fluorescence intensity of sample/
mean fluorescence intensity of the internal reference) × DNA
content of the internal reference (Kerker et al., 1982). Genome
size was estimated according to the conversion formula: 1 pg =
978 Mb (Dolezel and Bartos, 2005), and the mean of three
measurements was taken to determine the final genome size.
2.5 K-mer analysis and the estimation of
genome size
Genomic DNA was extracted from each sample using a modified
Cetyltrimethylammonium bromide (CTAB)–based method (Aboul-
Maaty and Oraby, 2019). Next, the DNA samples were randomly
fragmented using a Covaris ultrasonic disruptor to construct
sequencing libraries with fragment sizes of 150 bp. Subsequently,
high-throughput sequencing was performed using DNBSEQ-T7
platform. Raw reads were filtered by FastQC (v0.20.1) software to
remove low-density k-mers (<5) to minimize the impact of
sequencing errors, thus resulting in clean reads, which were then
used for subsequent analyses. To determine if there was any
contamination in the sequencing data, we extracted the first
50,000 reads from the sequencing data and performed a blast
alignment (v2.11.0+; parameters: evalue 1e-5 -max_target_seqs 1)
with the Nucleotide Transcript Database (NT Database) (v202107).
Species classification was conducted using MEGAN (v6.16.4).
Genome size was determined by GCE (v1.0.0.) This was
performed by determining the k-mer frequency-depth
distribution using Jellyfish (v2.2.10) and then estimating the
genome size based on the k-mer frequency-depth distribution
(Marçais and Kingsford, 2011). The genome size was calculated
in accordance with a previous study as genome size = total number
of k-mer/expected peak depth (Luo et al., 2023).
3 Results
3.1 Chromosome preparation and
karyotype analysis
3.1.1 Chromosome preparation
In this study, we used a range of biological specimens from H.
lycosina, including blood, legs, silk glands, gonads, and entire bodies,
to develop a chromosomal preparation method for the first time.
Our analysis demonstrated that metaphase chromosomes were
observed in the gonads with clear morphology but were either
not observed in the other tissues or there was evidence of
impurities, thus affecting observation (Supplementary Figure
S1E). The optimal metaphase morphology was achieved when
colchicine was used at a concentration of 0.05% and a treatment
duration of 2 h (Supplementary Figure S2C). The best chromosome
dispersion was achieved with 0.075 mol/L KCl solution for 1.5 h
(Supplementary Figure S3C). In addition, we found that the best
results were observed when samples were stained with 10% Giemsa
solution for 20 min (Supplementary Figure S4B). Therefore, this
optimized protocol was subsequently applied for chromosome
preparation in other species.
3.1.2 Karyotype analysis
This study is the first to reveal the chromosomal karyotypes of
two wolf spiders from the Lycosidae family with different habits. The
chromosome numbers were counted in female and male H. lycosina
had 26 and 24 chromosomes, with 13 and 12 pairs of chromosomes.
The female and male L. grahami were 20 and 18, paired with 10 and
9 pairs of chromosomes, respectively. Mitotic metaphase
chromosomes are depicted in Figure 2 (Full pictures are provided
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in Supplementary Figure S5). The principal characteristics of two
wolf spiders’chromosomal karyotypes were presented in Table 2,
with specific karyotypic parameters delineated in Supplementary
Table S2. In two wolf spiders, the female spiders possess two
additional chromosomes compared to the males, and both follow
the sexual chromosome system (SCS) X
1
X
2
O. The H. lycosina
chromosomes are predominantly metacentric (m) and
submetacentric chromosomes (sm) (Figures 2A,B). Conversely,
the L. grahami chromosomes are characterized mainly by the
presence of subtelocentric (st) and telocentric chromosomes (t)
(Figures 2C,D).
A comparison of the chromosome lengths between male and
female H. lycosina revealed that the mean length (7.69 μm) and
length range (6.22–9.32 μm) of female chromosomes were shorter
than those of males, which had a mean chromosome length of
8.33 μm and a length range of 5.61–10.63 μm). Comparison of the
arm ratio (the length of the long arm/short arm, AR) of female and
male chromosomes revealed that the mean AR (1.78) and AR range
(1.23–2.44) in males were greater than the mean AR (1.75) and AR
range (1.25–2.42) of females (Supplementary Table S2)). To
compare the length of each chromosome more intuitively, the
long arm and short arm of the chromosomes from male and
female spiders were presented as bar charts (Figures 3A,B). The
largest chromosomes in females and males were submetacentric
(sm) and metacentric chromosomes (m), with lengths of 9.32 μm
and 10.63 μm, respectively. The shortest chromosomes were all
metacentric chromosomes (m), with lengths of 6.22 μm in females
and 5.61 μm in males. In H. lycosina, karyotype asymmetry
coefficients (As.K%) were 63.01% in females and 63.28% in
males. The length ratios of the longest to shortest chromosomes
(L/S) were 1.50 in females and 1.90 in males. The proportion of
chromosomes with an arm ratio greater than 2:1 was 0.15 in females
and 0.13 in males. The karyotype classification for both female and
male H. lycosina was type 2A.
In a comparative study of the chromosome lengths in female and
male L. grahami, we observed that the mean length of chromosomes
in females (10.00 μm) was less than that in males (11.11 μm),
although the chromosome length range in females (6.91–13.74 μm)
was broader than that in males (8.30–14.19 μm). Comparison of the
AR of chromosomes in females and males revealed that the mean
AR (6.93) and range of AR (3.74–12.20), in males exceeded those in
females, which were 3.22 and 3.02–3.65, respectively
(Supplementary Table S2). For a more direct comparison of the
length of each chromosome, we generated bar graphs (Figures 3C,D)
to represent the long and short arms of the chromosomes in male
and female L. grahami. The largest and smallest chromosomes of
FIGURE 2
Metaphase mitotic chromosome (left) and karyotype arrangement diagrams (right) of two spiders. (A) Female H. lycosina (2n = 26). (B) Male H.
lycosina (2n = 24). (C) Female L. grahami (2n = 20). (D) Male L. grahami (2n = 18). Bar = 20 μm.
TABLE 2 Main karyotype characters of the two spiders.
Species CL (μm) ML (μm) MAR L/S As. K% KT KF
H. lycosina (\) 6.22–9.32 7.69 1.75 1.50 63.01 2A 14 m + 12 sm
H. lycosina (_) 5.61–10.63 8.33 1.78 1.90 63.28 2A 10 m + 14 sm
L. grahami (\) 6.91–13.74 10.00 3.22 1.99 96.30 4A 20st
L. grahami (_) 8.30–14.19 11.11 6.93 1.71 86.15 4A 12st+6t
CL, chromosome length range; ML, mean chromosome length; MAR, mean arm ratio; L/S: the ratio of the longest to the shortest chromosome length; As.K%, karyotype asymmetry coefficients;
KT, karyotype type; KF, karyotype formula.
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female and male spiders were subtelocentric chromosomes (st). The
lengths of the large chromosomes were 13.74 μm and 14.19 μm,
respectively, while the lengths of the small chromosomes were
6.91 μm and 8.30 μm, respectively. In L. grahami, females and
males exhibited As.K% values of 96.30% and 86.15%, respectively,
with L/S of 1.99 and 1.71. All chromosomes had arm ratios
exceeding 2:1. The karyotypes of both females and males were
classified as type 4A.
To demonstrate the existence of sex chromosomes in the
two spiders, we conducted meiotic observations on males from
each species. The analysis showed that in the meiotic cells of
both male species, the sex chromosomes could be readily
discerned during the pachytene nucleus due to their extreme
condensation condensation and positive heteropycnosis
(Figures 4A,B). Diakinesis cells confirmed the number of
bivalents and sex chromosomes in H. lycosina with
FIGURE 3
Karyotype diagrams for the two spiders. (A) Female H. lycosina (2n = 26, 14 m + 12 sm). (B) Male H. lycosina (2n = 24, 10 m + 14 sm). (C) Female
L. grahami (2n = 20, 20th). (D) Male L. grahami (2n = 18, 12th + 6t). Positive and negative values indicate short and long arms, respectively.
FIGURE 4
Meiotic cells in the two spiders, male. (A, C) H. lycosina.(B) L. grahami.(A, C) Cells from the two spiders in the pachytene stage of meiosis exhibited
positive heteropycnotic sex chromosomes. (C) Diakinesis cells confirmed the number of bivalents and sex chr omosomes in H. lycosina with 11 autosomal
bivalents and X
1
X
2
. The arrow represents the sex chromosome. Bar = 10 μm.
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11 autosomal bivalents and two sexual univalents (n = 11 +
X
1
X
2
)(Figure 4C).
3.2 Genome size analysis
3.2.1 Genome size estimation by flow cytometry
ThegenomesizesforthetwospiderswereestimatedbyFCM.
S. lycopersicum was used as an internal reference and T. clavata
was used as positive control. Three biological replicates of
measurements were performed for each species and results
exhibited good reproducibility. Analysis predicted that the size
of the T. clavata genome was 2720.15 Mb, which was consistent
with the genome assembly. The mean fluorescence intensities
were 6311.66 and 11,852.18 for H. lycosina and L. grahami,
respectively (Figure 5). The calculated genome sizes were
1966.54 Mb and 3692.81 Mb for H. lycosina and L. grahami,
respectively (Table 3).
FIGURE 5
Histogram shows 2C DNA content in the two spiders, as determined by flow cytometry. The X-axis represents the relative fluorescence intensity of
nuclei stained with PI in the nuclear suspension, while the Y-axis indicates the number of nuclei. (A) S. lycopersicum was used as the internal reference
(2C = 0.85 pg, 2C peak channel is 2668.08). (B) T. clavata (2720.15 Mb, 2C peak channel is 8730.39). (C) H. lycosina (1966.54 Mb, 2C peak channel is
6311.66). (D) L. grahami (3692.81 Mb, 2C peak channel is 11852.18).
TABLE 3 Genome sizes for the two spiders, as estimated by flow cytometry.
Species Fluorescence intensity of
internal reference
Fluorescence intensity
of sample
Fluorescence
intensity ratio
C-value/pg
DNA-2C
Genome
size (Mb)
H. lycosina 2668.08 6311.66 2.37 2.01 1966.54
L. grahami 2668.08 11,852.18 4.44 3.78 3692.81
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3.2.2 Genome size estimation by K-mer analysis
Next, we used the DNBSEQ-T7 high-throughput sequencing
platform to analyze H. lycosina and L. grahami using small insert
size library sequencing. After filtering low-quality sequencing
reads, the total number of bases in the H. lycosina and L. grahami
genomes was determined to be 454.88 Gb (Q30 95.40%) and
335.26 Gb (Q30 93.37%), with a GC content of approximately
32.45% and 32.17%, respectively (Supplementary Table S3).
Analysis of the distribution of GC content revealed that there
was no separation between AT and GC in the sequenced sequence
(Supplementary Figure S6). Next, 50,000 reads were extracted
from the filtered data and compared to the NT database
(Supplementary Table S4). Comparative analysis revealed that
there was no significant DNA contamination from external
sources. Collectively, our findings suggested that the
sequencing quality was excellent and could be used for survey
analysis as well as subsequent whole-genome sequencing.
Based on the 19-mer frequency distribution, the genome size
of H. lycosina was estimated to be 2099.89 Mb (Table 4), 1.07-
fold larger than that of the size estimated by FCM (1966.54 Mb);
the heterozygosity and duplication rates were 0.42% and
21.39%, respectively (Figure 6A). The estimated size of the
L. grahami genome was 4012.56 Mb (Table 4), 1.09-fold
larger than the size estimated by FCM (3692.81 Mb), with
heterozygosity and duplication rates of 0.80% and 54.91%
(Figure 6B). Both the rates of heterozygosity and duplication
in the L. grahami genome were larger than those in the H.
lycosina genome.
4 Discussion
4.1 Chromosome preparation
High-quality mitosis metaphase cells are essential for
chromosome karyotype analysis. In the field of spider
chromosome research, seven methods for preparing chromosome
slides of spiders have been documented and experimentally verified:
reproductive gland section, testis squash, single embryo drop, mixed
embryo drop, blood cell preparation, single embryo squash, and
needle smear technique. However, these methods have many
drawbacks in practical application. The reproductive gland
section technique has a low detection rate, and there are
variations in chromosome morphology and number, which is
now rarely used. The testis squash technique is currently mainly
used for preparing meiotic chromosome spreads (Král et al., 2022).
The single embryo drop technique generally yields unsatisfactory
results (Matsumoto, 1977). The mixed embryo drop technique
yields better results, but it is prone to chromosome loss during
staining, and the significant differences in oviposition times among
different spiders make it difficult to collect materials (Hu et al.,
2023). The blood cell chromosome preparation method yields poor
results, as there are large individual variations in spider size, and
spider blood is scarce, making it difficult to collect materials. The
needle smear method involves feeding spiders with colchicine and
then preparing slides from the gonads, which yields relatively ideal
results, but the experimental cycle is relatively long, making it
difficult to control variables. Therefore, in this study, we
TABLE 4 19-mer analysis for the estimation of genome size in the two spiders.
Sample K-mer number K-mer depth Genome size (Mb) Heterozygous ratio (%) Duplication ratio (%)
H. lycosina 340,518,852,100 161.79 2099.89 0.42 21.39
L. grahami 247,773,629,878 61.68 4012.56 0.80 54.91
FIGURE 6
19-mer frequency distribution graph of sequencing reads for the two spiders. The X-axis represents the sequencing depth, while the Y-axis
represents the ratio of specific k-mer to the total number of k-mer for a given sequencing depth. (A) Distribution curve of H. lycosina.(B) Distribution
curve of L. grahami.
Frontiers in Genetics frontiersin.org08
Zhang et al. 10.3389/fgene.2025.1544087
optimized chromosome preparation methods for two species of
spiders, including sampling, pretreatment, and hypotonic treatment
steps, and successfully generated metaphase chromosomes for
karyotype analysis. Compared to traditional methods, the
approaches used in this study are more straightforward to
operate and offer better reproducibility. Tissues with high mitotic
activity are preferred for karyotype preparations as these ensure a
sufficient number of mitotic phases (Indy et al., 2010).
In Araneae, various tissues have been found to be suitable for
chromosome preparation, including embryos, blood, abdominal
tissues, and gonads (Wayne et al., 2020). In the present study, we
identified gonads as the optimal tissue for metaphase spread
acquisition, as other tissues yielded fewer mitotic phases,
potentially due to a lower mitotic index. Furthermore,
pretreatment is a crucial step when generating chromosome
preparations; consequently, the dose of colchicine and the
processing time are important factors that can affect
chromosome preparation. The role of this treatment is to impede
the formation of the spindle, prompting the chromosomes to
shorten and thicken, thus generating metaphase spreads with
suitable chromosome morphology (Tianwen et al., 2020).
Furthermore, the complete dissection of gonads is known to have
a significant impact on chromosome preparation.
4.2 Karyotype analysis
In this research, we conducted comparative karyotype analysis
of H. lycosina and L. grahami, two species of spider from Yunnan,
China. The analysis revealed that the chromosome number of
diploids and SCS observed in the two spiders were consistent
with those previously reported for the congeneric species H.
agelenoides and Lycosa sp.: 2n_= 24 and 2n_= 18, X
1
X
2
O-X
1
X
2
(Sharma et al., 1958;Srivastava and Shukla, 1986). The Lycosidae
family exhibits considerable diversity in chromosome number, with
male diploid numbers (2n) ranging from 18 (in L. narborensis,
Lycosa sp., and this study) to 28 (in Alopecosa aculeata,L. bistriata,
and Arctosa cinerea, among others) (Supplementary Table S1).
Notably, a chromosome number of 28 has been detected in over
50% of species; this potentially represents the modal diploid number
within the family (Chemisquy et al., 2008). However, many
Lycosidae species still exhibit lower diploid numbers (2n_<28)
(Supplementary Table S1, S5, and this study). It is worth noting that
a chromosome number of 18 is the lowest ever reported in the family
Lycosidae and has only been only reported in the genera Lycosa.
Previous studies have hypothesized that the ancestral male
karyotype of wolf spiders consists of 28 acrocentric chromosomes
(Bugayong et al., 1999;Datta and Chatterjee, 1989;Dolejšet al.,
2011;Sharma et al., 1958). In some Lycosidae lineages, a gradual
reduction in diploid chromosome number has occurred, likely due
to centromere fusion, a phenomenon that is also observed in other
entelegyne spiders (Suzuki, 1954). Except for the ancestral number
of 2n = 28, chromosome number reductions are particularly evident
in the Lycosinae, Evippinae, and Venoniinae subfamilies (Dolejš
et al., 2011).
We identified significant differences in chromosome number
and morphology between the two spiders, indicating that these two
species possess distinct genetic characteristics. In addition, a
chromosomal discrepancy existed between the sexes of these
spiders. We found that males had two fewer chromosomes than
females. This disparity, along with variations in karyotypic formulae,
morphology, and size, may correlate with SCS differences. In the
Lycosidae family, the SCS has been classified as XO (Araujo et al.,
2015), X
1
X
2
O, and X
1
X
2
X
3
O(Brum-Zorrilla and Postiglioni, 1980).
Our study confirmed that both species were X
1
X
2
O SCS, which is
prevalent among the Lycosidae. Typically, 95% of Lycosidae species
have an SCS of X
1
X
2
O-X
1
X
2
/X
1
X
1
X
2
X
2
(male/female), in which O
indicates the absence of a Y chromosome, and the male sex
chromosome consists of two X chromosomes and the female sex
chromosome consists of two pairs of X chromosomes (Chemisquy
et al., 2008). This SCS is the ancestral type of spiders including the
Lycosidae (Datta and Chatterjee, 1989;Dolejšet al., 2011;Suzuki,
1954). However, sex chromosomes varied in length among the
species of Lycosidae studied thus far, including the largest,
medium-sized, and smallest length of chromosomes (Araujo
et al., 2015;Cavenagh et al., 2022;Dolejšet al., 2011). These
differences may be due to sex chromosome and/or autosomal
rearrangements that result in variations in their size. Regrettably,
our study could only confirm the presence of two sex chromosomes
in male specimens during specific meiotic phases, making it
impossible to determine the exact location and size of the sex
chromosomes.
4.3 Genome size analysis
ThegenomeofonlyspeciesfromtheLycosidae,Pardosa
pseudoannulata, has been reported previously; this genome was
2420 Mb in size and had high levels of heterozygosity (2.77%) (Yu
et al., 2024).ThesizeoftheH. lycosina genome generated in this
study was 1966.54–2099.89 Mb and had low levels of
heterozygosity and repetition. In addition, the size of the
L. grahami genome was 3692.81–4012.56 Mb and had higher
levels of heterozygosity and repetition. In addition, we also found
that the genome size predicted by flow cytometry was slightly
lower than that predicted by the genome k-mer analysis. This
may have been caused by the flow cytometry technique being
affected by factors such as sampling site, internal reference
selection, and the processing environment when determining
genome size (Dai et al., 2022). Of the two methods used to
determinegenomesizeinthisstudy,flow cytometry is by far
the most commonly used method, predominantly because it is
low cost and fast, but this method also requires expensive
instruments and internal references (Del Mar Ochoa-Saloma
et al., 2020;He et al., 2016;Liu et al., 2016;Pflug et al., 2020;
Swathi et al., 2018).TheselectingoftheplantS. lycopersicum as
an internal reference in this study is based on its stable and well-
defined genome size, which has been widely used in flow
cytometry studies (Cerca et al., 2022). To further verify the
reliability of S. lycopersicum as an internal reference, this
study also employed T. clavata, a species with a well-
characterized genome size, as a positive control (Hu et al.,
2023). The result indicated that the flow cytometry-predicted
genome size of T. clavata (2720.15 Mb) was essentially consistent
with the published value. It suggests that S. lycopersicum can
serve as an effective internal reference species for predicting the
Frontiers in Genetics frontiersin.org09
Zhang et al. 10.3389/fgene.2025.1544087
genomesizesofthetwowolfspiders.K-meranalysiscanyield
rich and accurate information and can predict genome size,
heterozygosity, the repetitive sequence ratio, and GC content.
However, it can be easily affected by multiple factors, such as data
quality, the selected software, and the parameter settings (Baeza
et al., 2021;Fan et al., 2022). Therefore, these methods are often
used in combination to provide a comprehensive judgment when
estimating the genome size of a species within a relatively
accurate range (Luo et al., 2023;Mgwatyu et al., 2020;Zhou
et al., 2023).
Generally, higher-level organisms possess more complex
genetic information than lower-level organisms, which might
suggest that higher-level organisms have larger genome sizes.
However, there is no strict correlation between genome size and
the complexity of organisms (the C-value paradox) (Kim et al.,
2014). This is because genomes contain many highly repetitive
DNA sequences, resulting in a conflict between DNA content and
evolutionary level. In a previous study of the Araneae, the
genome sizes of 26 spiders were determined by FCM, ranging
from 1.7 to 4.7 Gb (Král et al., 2019). In contrast, the genome sizes
of spiders predicted by k-mer analysis ranged from 0.7 Gb in O.
gibbosus to 6.50 Gb in Acanthoscurria geniculata (Sanggaard
et al., 2014;Wang et al., 2022). These previous studies
highlight the substantial variation in DNA content even
among closely related species. Spiders, as an ancient and
highly diverse group of animals, exhibit considerable variation
in genome size and karyotype. These differences may be closely
related to the different ecological habits, living environments,
and evolutionary histories of the species (Lertzman-Lepofsky
et al., 2019;Liedtke et al., 2018;Saha et al., 2023).
To investigate the potential link between the genome sizes
and karyotypes of spiders, we collected existing data relating to
thegenomesizeandkaryotypeof16spiderspeciesand
performed a comparative analysis. The results revealed
significant differences in genome size and chromosome
number both among different families and within the same
family. Moreover, no significant correlation was found
between genome size and chromosome number
(Supplementary Figure S7). A previous study detected
chromosome numbers in 6,052 genome size records and found
no significant correlation between genome size and chromosome
number (El Shehawi and Elseehy, 2017). However, due to the
limited sample size in our study, which only investigated the
karyotypes and genome sizes of two spider species, the
generalizability of our findings is constrained. Consequently,
further research and validation are essential to better
understand the diversity and evolutionary history of
Lycosidae species.
This study was the first preliminary study exploring the
karyotype and genome size of two wolf spiders with distinct
habits, both belonging to the Lycosidae. We found that these
spiders exhibited significant differences in genome sizes,
chromosome numbers, morphologies, and sizes, potentially
reflecting their unique genetic traits and evolutionary
histories. Karyotype analysis revealed that the webbing H.
lycosina primarily has metacentric and submetacentric
chromosomes, while the hunting L. grahami features
predominantly acrocentric and telocentric chromosomes; the
SCS was X
1
X
2
O. The genome of H. lycosina was relatively
small, with low levels of heterozygosity and repetition, while
the L. grahami genomewaslargerwithhigherlevelsof
heterozygosity and repetition. Collectively, our findings
provide novel insights into the karyotype, genome size,
heterozygosity, and repetitive sequence proportions of the two
spiders, yielding valuable data for further research in
cytogenetics, taxonomy, phylogeny, and whole-genome
sequencing of Lycosidae.
Data availability statement
The data used in this study can be found in the article/
Supplementary Materials.
Author contributions
YZ: Data curation, Investigation, Methodology, Software,
Visualization, Writing–original draft, Writing–review and editing.
MZ: Methodology, Resources, Writing–original draft. LL:
Conceptualization, Writing–review and editing. YW:
Conceptualization, Writing–review and editing. HY:
Conceptualization, Writing–review and editing. LW:
Conceptualization, Writing–review and editing. BL:
Conceptualization, Writing–review and editing. SY:
Conceptualization, Writing–review and editing. ZY: Funding
acquisition, Resources, Writing–review and editing. SC: Funding
acquisition, Writing–review and editing. CS: Funding acquisition,
Writing–review and editing.
Funding
The author(s) declare that financial support was received for
the research and/or publication of this article. This research was
supported by “Introduces the talented person scientific research start
funds subsidization project”of Chengdu University of Traditional
Chinese Medicine (Reference: 030040015 and 030040017).
Acknowledgments
The authors are grateful to Wuhan Benagen Technology
Company for their assistance with sequencing and
bioinformatics analysis.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Frontiers in Genetics frontiersin.org10
Zhang et al. 10.3389/fgene.2025.1544087
Generative AI statement
The authors declare that no Generative AI was used in the
creation of this manuscript.
Publisher’s note
All claims expressed in this article are solely those of the authors and
do not necessarily represent those of their affiliated organizations, or
those of the publisher, the editors and the reviewers. Any product that
may be evaluated in this article, or claim that may be made by its
manufacturer, is not guaranteed or endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fgene.2025.1544087/
full#supplementary-material
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