Different Chromosome Segregation Patterns Coexist in the Tetraploid Adriatic Sturgeon Acipenser naccarii

Abstract

The Adriatic sturgeon, Acipenser naccarii (Bonaparte, 1836), is a critically endangered tetraploid endemism of the Adriatic region; it has been targeted, over the last 20 years, by different conservation programs based on controlled reproduction of captive breeders followed by the release of their juvenile offspring; its preservation would greatly benefit from the correct and coordinated management of the residual genetic variability available in the different captive stocks. In this sense, the setup of an efficient parental allocation procedure would allow identifying familiar groups and establishing informed breeding plans, effectively preserving genetic variation. However, being the species tetraploid, the analyses often deal with complex genome architecture and a preliminary evaluation of allele segregation patterns at different chromosomes is necessary to assess whether the species can be considered a pure tetraploid, as previously observed at some loci, or if a more complex situation is present. Here we study the segregation at 14 microsatellites loci in 12 familiar groups. Results support in different families the tetrasomic segregation pattern at 11 markers and the disomic segregation at three markers. The Adriatic sturgeon thus shows a mixed inheritance modality. In this species, and likely in other sturgeons, accurate knowledge of the loci used for paternity analysis is therefore required.

Full text

Diversity 2022, 14, 745. https://doi.org/10.3390/d14090745 www.mdpi.com/journal/diversity
Article
Different Chromosome Segregation Patterns Coexist in the
Tetraploid Adriatic Sturgeon Acipenser naccarii
Stefano Dalle Palle
1,†
, Elisa Boscari
1,†
, Simone Giulio Bordignon
1
, Víctor Hugo Muñoz-Mora
2
,
Giorgio Bertorelle
2
and Leonardo Congiu
1,
*
1
Department of Biology, University of Padova, Via Ugo Bassi 58b, 35121 Padova, Italy
2
Department of Life Sciences and Biotechnology, University of Ferrara, Via Luigi Borsari 46, 44121 Ferrara,
Italy
* Correspondence: leonardo.congiu@unipd.it
† These authors contributed equally to this work.
Abstract: The Adriatic sturgeon, Acipenser naccarii (Bonaparte, 1836), is a critically endangered
tetraploid endemism of the Adriatic region; it has been targeted, over the last 20 years, by different
conservation programs based on controlled reproduction of captive breeders followed by the release
of their juvenile offspring; its preservation would greatly benefit from the correct and coordinated
management of the residual genetic variability available in the different captive stocks. In this sense,
the setup of an efficient parental allocation procedure would allow identifying familiar groups and
establishing informed breeding plans, effectively preserving genetic variation. However, being the
species tetraploid, the analyses often deal with complex genome architecture and a preliminary
evaluation of allele segregation patterns at different chromosomes is necessary to assess whether
the species can be considered a pure tetraploid, as previously observed at some loci, or if a more
complex situation is present. Here we study the segregation at 14 microsatellites loci in 12 familiar
groups. Results support in different families the tetrasomic segregation pattern at 11 markers and
the disomic segregation at three markers. The Adriatic sturgeon thus shows a mixed inheritance
modality. In this species, and likely in other sturgeons, accurate knowledge of the loci used for
paternity analysis is therefore required.
Keywords: acipensaeridae; autotertraploid; allopolyploid; Adriatic sturgeon; disomic segregation;
inheritance; microsatellites; tetrasomic segregation
1. Introduction
Sturgeons are the most endangered group of species, according to the International
Union for Conservation of Nature (IUCN, July 2021) (http://www.iucnredlist.org,
accessed on 8 August 2022). For this reason, sturgeons are targeted by several
conservation efforts that often include restocking programs with juveniles produced in
captivity; these ex-situ conservation activities must necessarily be supported by studies
aimed at preserving the residual genetic diversity through long-term breeding programs
[1]. However, genetic analyses on sturgeons deal with complex genomes and various
levels of ploidy, due to independent events of whole-genome duplication [2,3]. The first
event of duplication took place in the Acipenseriformes’ common ancestor starting from
sixty chromosomes. Then, secondary events of duplication occurred in the Pacific and
Atlantic clades leading to a total of 240 chromosomes [2]. Finally, a third event led to the
unique number of 360 chromosomes observable in Acipenser brevirostrum (Lesueur, 1818)
[4]. The number of chromosomes associated with the distinct levels of ploidy has been the
subject of an extensive debate between two main positions. The first argues that, since the
condition with 120 chromosomes results from a duplication event in the common
ancestor, the species with 120 and 240 chromosomes must be considered tetraploid and
Citation: Dalle Palle, S.; Boscari, E.;
Bordignon, S.G.; Muñoz-Mora, V.H.;
Bertorelle, G.; Congiu, L. Different
Chromosome Segregation Patterns
Coexist in the Tetraploid Adriatic
Sturgeon Acipenser naccarii. Diversity
2022, 14, 745. https://doi.org/10.3390/
d14090745
Academic Editors: Michael Wink
and Simon Blanchet
Received: 13 August 2022
Accepted: 7 September 2022
Published: 10 September 2022
Publisher’s Note: MDPI stays
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Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
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Attribution (CC BY) license
(https://creativecommons.org/license
s/by/4.0/).

Diversity 2022, 14, 745 2 of 17
octaploid, respectively. The second position, taking into account the functional reduction
of ploidy that follows whole genome duplications, attributes to the two groups a condition
of diploidy and tetraploidy, respectively [5]. The two views use different criteria to define
the nominal ploidy, the number of duplications and the functional activity of genes,
respectively, and are both correct and fully compatible [3].
The Adriatic sturgeon, Acipenser naccarii (Bonaparte, 1836), a species with 240
chromosomes, is in general considered a functional tetraploid based on the analyses of
28S and 5S rDNA through in situ hybridization [6]; this is also confirmed by most of the
microsatellites analysed in this species which mostly show up to 4 alleles per individuals.
However, some loci consistently showed more than 4 alleles in many individuals [7]; this
could be due to the duplication of the region of such microsatellites but, a still incomplete
process of functional reduction following genome duplication cannot be excluded as
already proposed also for other sturgeon species [8,9].
For these reasons, loci to be used for applied purposes, such as parental allocation
and kinship analysis, should be carefully selected and their functional ploidy should be
preliminarily assessed. Moreover, these applications require a careful preliminary
investigation also on the segregation modalities which, in tetraploids, can be of different
types. In fact, polyploidization can originate through the fusion of unreduced gametes at
intraspecific or interspecific levels, leading to two types of conditions in tetraploids, called
autotetraploidy and allotetraploidy, respectively. The origins of polyploidization have
important implications on the segregation patterns of the alleles within gametes. In
complete autotetraploids, there are always four homologous chromosomes, and random
pairs of bivalents and quadrivalents are possible during meiosis (see Figure 1 for graphical
support) [10]; this condition leads to tetrasomic inheritance, which means that all allelic
combinations within gametes are possible; this is not the case in complete allotetraploids,
where each tetrad is composed of two sets of homeologous chromosomes, originating
from the two parental species. In this situation, the homeologous chromosomes do not
form pairs, leading to disomic inheritance, where only four out of six allelic combinations
are possible [10]; these are two extreme cases and intermediate conditions are observable
when the chromosomes have different degrees of preferential pairing. Indeed, the
inheritance may shift from tetrasomic to disomic or vice versa [10]. For example, in
autotetraploids, fertility and karyotype stability can be negatively impacted by imperfect
multivalent pairing, thus promoting diploidization and consequent shifting to disomy; on
the contrary, in allotetraploids, the homeologous chromosomes from two distinct parental
species could maintain some degree of genetic affinity permitting the competition with
the homologous pair during meiotic interactions with a certain degree of tetrasomic
inheritance [11]. Understanding the mechanisms of chromosomal segregation in a
tetraploid species can have important conservation implications, for example when the
development of parental allocation methods is required.

Diversity 2022, 14, 745 3 of 17
Figure 1. Expected segregation patterns under pure disomy (a) or tetrasomy (b), respectively
expected in Autotetraploid and Allotetraploid genomes.
In the Adriatic sturgeon, allele segregation was previously investigated at only 7 loci
using microsatellite markers [12]. All loci showed a tetrasomic inheritance pattern,
pointing at the probable autopolyploidization origin of this species. However, as
secondary differentiation of some homologous chromosomes cannot be excluded and
different segregation patterns can be followed by different chromosomes, we took
advantage of the recent availability of new complete family groups and new isolated and
tested microsatellite loci not yet explored, to provide a deeper insight into the mode of
chromosome segregation in this species.
This is a key step not only to have a better understanding of the karyotype in the
Adriatic sturgeon but also for the correct interpretation of the genetic analysis and
parental allocation which in this species can have multiple applications. Firstly, the
distinction of extremely rare individuals of wild origin from released ones. Secondly, the
identification of groups of siblings existing in the different farms by assigning everyone
to his pair of parents of the F0 generation. Finally, knowing the segregation patterns is
crucial when the generation of virtual genotypes starting from observed genetic profiles
is needed [13,14].
Thanks to the possibility of making 12 distinct crosses and raising their progeny
separately within the project ENDEMIXIT (https://endemixit.com/, accessed on 8 August
2022), many new informative familiar groups became available for the analysis of
segregation patterns at a much higher number of loci than those available in the past. The
main purpose of the present study is therefore to exhaustively describe the modalities of
microsatellite alleles inheritance in the Adriatic sturgeon with the following objectives: (a)
verifying if the inferred pure tetrasomy is confirmed on a high number of loci, (b)
providing a significant contribution to the management of the residua genetic diversity of
this critically endangered species, and (c) shedding light on the functional ploidy level
across its genome.
2. Materials and Methods
2.1. Samples and DNA Purification
Most samples analyzed in the present study were obtained from the aquaculture
plant Storione Ticino (Cassolnovo, Italy). The reproduction of six females (F2, F4, F5, F6,
F7, and F8) and six males (M2, M4, M5, M6, M7, and M8) was performed in different

Diversity 2022, 14, 745 4 of 17
combinations. For each parent, a fin clip was collected for genotyping and, for each cross,
the progeny was reared in captivity and spontaneously dead animals were collected and
stored in ethanol. Moreover, five familiar groups (Nacc7 ♀ ×Nacc5 ♂, Nacc8 ♀ × Nacc31 ♂,
Nacc19 ♀ × Nacc17 ♂, Nacc19 ♀ × Nacc30 ♂, Nacc28 ♀ × Nacc23 ♂) derived from crosses
performed in the past and for which fingerlings were available were also used. In total, 21
adult parents (12 parent pairs) and 376 fingerlings (with about 30 individuals per family)
were collected (Table 1).
Table 1. Screening of microsatellite loci.
Locus Ref Ta Size Family Parents Genotypes Genotyped Fingerling (Y/N) #
LS-39 15 52 °C 116–155 Not informative - - N -
AfuG113 16 Td 326–364 Not informative - - N -
AfuG132 16 61 °C 259–346
F5 ♀ × M6 ♂ F5 ♀
M6 ♂
293/309/318/330
313/318/322 Y 32
F6 ♀ × M7 ♂ F6 ♀
M7 ♂
313/318/342
293/309/318/322 Y 32
F8 ♀ × M2 ♂ F8 ♀
M2 ♂
304/318/322
313/338/342/346 Y 30
AfuG41 16 58 °C 156–198
F8 ♀ × M2 ♂ F8 ♀
M2 ♂
212/234/293
203/255/259/285 Y 30
F2 ♀ ×M4 ♂ F2 ♀
M4 ♂
247/255/259/285
212/255/293/305 Y 30
F4 ♀ ×M4 ♂ F4 ♀
M4 ♂
212/217/242/259
212/255/293/305 Y 30
F6 ♀ ×M7 ♂ F6 ♀
M7 ♂
212/242/259/278
203/225/229/259 Y 32
Nacc7 ♀ ×Nacc5 ♂ Nacc7 ♀
Nacc5 ♂
225/229/252/259
203/225/247/305 Y 32
Nacc8♀ ×Nacc 31♂ Nacc8 ♀
Nacc31 ♂
212/242/293/305
203/234/305 Y 32
An20 * 17 62 °C 159–213 F7 ♀ ×M8 ♂ F7 ♀
M8 ♂
160/164/194
160/172/182/186 Y 30
Anac-15214 7 61 °C 259–285 Not informative - - N -
Anac-2589 7 63 °C 224–294 Not informative - - N -
Anac-6784 7 62 °C 311–346
Nacc19 ♀ × Nacc17 ♂ Nacc19 ♀
Nacc17 ♂
311/322/330/334
322/326 Y 32
Anac-3133 7 56 °C 164–178 F7 ♀ × M8 ♂ F7 ♀
M8 ♂
164/174
164/166/170/172 Y 30
AnacA6 * 18 62 °C 289–313 F5 ♀ × M6 ♂ F5 ♀
M6 ♂
307/313
293/297/301/307 Y 32
AnacB11 18 60 °C 132–162
F6 ♀ × M7 ♂ F6 ♀
M7 ♂
148/150
138/144/148/162 Y 32
Nacc19 ♀ × Nacc17 ♂ Nacc19 ♀
Nacc17 ♂
132/136/138/144
132/150/162 Y 32
AnacB7 18 60 °C 152–198
F6 ♀ × M7 ♂ F6 ♀
M7 ♂
166/172/174/176
154/156/164 Y 32
Nacc19 ♀ × Nacc30 ♂ Nacc19 ♀
Nacc30 ♂
156/166/174
154/170/174/176 Y 32
AnacC11 18 50 °C 167–193 Not informative - - N -
AnacE4 * 18 58 °C 326–354 F5 ♀ × M6 ♂ F5 ♀ 332/340/346/354 Y 32

Diversity 2022, 14, 745 5 of 17
M6 ♂ 336/346
AoxD161 19 60 °C 111–155
F4 ♀ × M5 ♂ F4 ♀
M5 ♂
123/127/131/139
131/135/155 Y 30
F8 ♀ × M2 ♂ F8 ♀
M2 ♂
123/131/135/139
127/131 Y 30
Nacc7 ♀ × Nacc5 ♂ Nacc7 ♀
Nacc5 ♂
123/127/131/143
131/135/139 Y 32
AoxD234 * 19 52 °C 215–275
F2 ♀ × M4 ♂ F2 ♀
M4 ♂
219/223/243/255
227/243 Y 30
F4 ♀ × M5 ♂ F4 ♀
M5 ♂
227/243/247/251
235/239/255/259 Y 30
Nacc28 ♀ × Nacc23 ♂ Nacc28 ♀
Nacc23 ♂
219/243/247/263
239/243/251/255 Y 24
F6 ♀ × M7 ♂ F6 ♀
M7 ♂
227/243/247/255
219/239/247 Y 32
AoxD241 * 19 57 °C 156–196 F7 ♀ × M8 ♂ F7 ♀
M8 ♂
168/176/180
164/172/176/184 Y 30
AoxD64 * 19 60 °C 216–260 Not informative - - N -
Spl-120 20 55 °C 263–303 Not informative - - N -
Spl-163 20 63 °C 166–233 F2 ♀ × M4 ♂ F2 ♀
M4 ♂
207/215/220
166/215/224/229 Y 30
Spl-168 10 63 °C 200–314
F7 ♀ × M8 ♂ F7 ♀
M8 ♂
232/240/294
218/236/271/273 Y 30
F4 ♀ × M5 ♂ F4 ♀
M5 ♂
209/232/257/273
218/232/279/282 Y 30
F6 ♀ × M7 ♂ F6 ♀
M7 ♂
227/236/271/286
240/245/264/269 Y 32
Nacc19 ♀ × Nacc17 ♂ Nacc19 ♀
Nacc17 ♂
218/227/269/294
227/240/286/314 Y 32
Nacc7 ♀ × Nacc5 ♂ Nacc7 ♀
Nacc5 ♂
232/245/264/294
214/240/273/279 Y 32
Nacc8 ♀ × Nacc31 ♂ Nacc8 ♀
Nacc31 ♂
209/236/271/279
218/249/273/279 Y 32
Loci were used for genotyping of breeders for the selection of informative family/locus combina-
tions for which progeny was also processed. References, annealing temperatures used, and size are
reported. For each informative family/locus combination, the genotypes of each parent at such loci
are also reported and the parent’s genotypes for which the allele segregation was followed in the
progeny is in bold. The total number of fingerlings processed for each informative family is reported
in the last column. * Loci for which the segregation inheritance pattern was already explored in
other familiar groups of the same species [12]. Y = Yes, N = Not processed. # = number of analyzed
individuals.
Genomic DNA was extracted from breeder’s fin clips (10–100 mg) and from off-
spring’s muscular tissue, using Euroclone spinNAker Universal Genomic DNA mini kit
(Euroclone) and stored at −20 °C till their processing for microsatellite analysis.
2.2. Selection of Loci and Genotyping
Loci analysed in the present study were selected based on the possibility of unam-
biguously tracing the genetic contribution of at least one of the two parents in at least one
of the available families. For this reason, all loci in which there were no complete hetero-
zygotes or there were individuals with more than four alleles were discarded. In fact, it is

Diversity 2022, 14, 745 6 of 17
known that the Adriatic sturgeon presents a minority of loci at which more than four al-
leles were can be observed in different individuals; thus, segregation anomalies cannot be
detected [7]; these extra numerary alleles could originate from duplication or from a lo-
cally unreduced octaploid condition. In fact, we recall that the Adriatic sturgeon is func-
tionally a tetraploid but evolutionarily it is an octaploid [3]. Nevertheless, segregation
anomalies were also observed in some individuals of each family that were accordingly
excluded from the analysis.
All breeders were genotyped at 21 microsatellite loci (Table 1) [7,15–20] to select the
informative Family/Locus combinations for the assessment of chromosomal segregation.
For each selected combination, the progeny was also amplified and genotyped. Tracking
segregation in the progeny requires the satisfaction of some features, such as (i) the com-
plete heterozygosity (four different alleles) of at least one parent to ensure that each allele
can unambiguously mark the segregation of its own chromosome and (ii) no more than
an allele shared by the two parents to avoid ambiguity in following the alleles transmis-
sion to the progeny [12].
A total of 33 Family/Locus combinations were finally selected and approximately 30
fingerlings each were genotyped (Table 1). In 22 out of 33 case studies only one breeder
of the parent pair was completely heterozygote and therefore informative to follow the
segregation of the alleles, while for the remnant 11 Family/Locus combinations the segre-
gation pattern was followed for both breeders of the parent pair as both they showed
completely heterozygous genotypes with a single allele shared at most (Table 1).
Microsatellite loci were amplified from genomic DNA in a 10 µL reaction: 1X Master
Mix Buffer (QIAGEN), 0.2 µM of each primer and about 50 ng of genomic DNA. Ampli-
fications were performed on SimpliAmp Thermal Cycler (Thermo Fisher Scientific, Bolo-
gna, Italy). Amplifications were checked on 1.8% agarose gel in TBE1X stained with
GelRed (BIOTIUM, Fremont, Canada, GelRed™ Nucleic Acid Stain). Genotyping was per-
formed on ABI PRISM 310 Genetic Analyzer (external service, BMR Genomics, Padova,
Italy). For microsatellite scoring the software GeneMarker Version 1.95 (SoftGenetics LLC,
State College, PA, USA) was used.
2.3. Data Analyses
Scoring was performed by two operators, independently, with GeneMarker Version
1.95 (SoftGenetics LLC). The final dataset was carefully checked, and each individual was
controlled for perfect Mendelian inheritance within his Family/Locus combination under
the assumption that the observed genotypes at a given locus should be compatible with
the inheritance of two allele copies from each parent. Individuals that for different possi-
ble reasons discussed later did not match this assumption were discarded. Since discord-
ances with the above-accepted criterion can be attributed to anomalies in chromosomal
segregation in the gametes of one parent, which are independent of what happened in the
mating partner’s gametes, it was decided not to discard those animals whose segregation
anomaly was due to a problem in the uninformative parent. Limiting our observations to
those animals that met standard inheritance of two out of four alleles, we consciously ex-
cluded possible genotypes that were not necessarily derived from segregation anomalies
such as the case of double reduction, a phenomenon widely observed in autotetraploid
plants in which the four homolog chromosomes may form multivalents [21,22] in which
identical alleles carried on the sister chromatids may enter the same gamete with conse-
quent segregation of two copies of the same allele even if it was present in a single copy
in the parental genome [23]. We have decided to limit ambiguities by disregarding this
phenomenon and excluding all the individuals that could be compatible with it focusing
on animals in which alleles were unambiguously traceable.
As proposed by Stift et al. (2008) [10], Likelihood Ratio Tests (LRT) with 1 df were
applied to compare the null model of tetrasomy with the other alternative models inter-
mediate between disomic and tetrasomic. For each parent-locus combination and each
alternative inheritance model, the log-likelihood was estimated from constrained

Diversity 2022, 14, 745 7 of 17
nonlinear regression models, using SPSS syntax as reported in the original reference [10].
The Sequential Bonferroni correction [24] was applied to adjust significance levels (p <
0.05) for multiple comparisons across loci and families.
3. Results
From 22 to 32 individuals for each family-locus combination were successfully gen-
otyped. In some cases, a few animals were discarded for unreliable profiles or, after the
allele scoring, due to the presence of segregation anomalies of alleles inherited from the
informative parent (Table 2).
At 11 (AfuG132, AfuG41, An20, Anac6784, Anac3133, AnacA6, AnacB11, AnacB7,
AnacE4, AoxD234, AoxD241) out of 14 informative loci the LRT did not lead to the rejec-
tion of the null model of inheritance thus supporting the hypothesis of tetrasomy. At al-
most all these loci, indeed, all six allele combinations were observed in almost all tested
families (Figure 1a, Table 2, Figure A1 of Appendix A). The only exceptions were the loci
AoxD234 and Anac-3133 at a single family each showing only five allele combinations.
However, in both cases, the strict disomic inheritance was excluded after correction for
multiple tests (Table 2). In these two families, the sixth combination is expected to be de-
tectable by increasing the sample size. Five (An20, AnacA6, AnacE4, AoxD234, and
AoxD241) of the 11 loci showing tetrasomic inheritance were already tested to assess the
inheritance pattern in different familiar groups of Adriatic sturgeon [12] and their tetra-
somy has been here confirmed. Specifically, the locus AnacA6 here analysed at a single
family showed only four allele combinations but, missing combinations are not compati-
ble with disomic inheritance modality and even in this case the null model was not re-
jected.
On the contrary, a significant rejection of the null model was observed at three loci,
Spl163, AoxD161, and Spl168, analysed respectively at one, three and six families and
never analysed before in other studies. In some cases, the analysed families have both
parents informative for segregation and agree in suggesting a disomic mode of inheritance
(Table 2, Figure 1b, Figure A1 Appendix A). The disomic model fitted the observed allele
combination frequencies significantly better than the null model, and the parameter τ
equal to zero indicates a full disomic inheritance at almost all Family/Locus combinations.
The only two exceptions were observed in the segregation of the alleles of female F8 at
locus AoxD161 and male Nacc31 at locus Spl168 (Table 2). In these two cases, five combi-
nations of alleles were present, the parameter τ assumed a very low value confirming a
strong degree of preferential pairing during meiosis but the rejection of the null model
was not significant after the Bonferroni correction, thus suggesting possible imperfect
preferential pairing.
Table 2. Likelihood Ratio Test.
Locus Family
Informative
Parent
N
(Nd)
Null Model (Τ = 1) Like-
lihood Obs
Best Intermediate Model Model Compar-
ison: LRT p-Values
Pairing
Alleles Τ Likelih-ood
Obs
Afug132
F5 ♀ × M6 ♂ F5 ♀ 22 (8) 39.42 AC/BD 0.82 39.23 0.19 0.3322
F6 ♀ × M7 ♂ M7 ♂ 29 (1) 51.96 AD/BC 0.83 51.74 0.22 0.3185
F8 ♀ × M2 ♂ M2 ♂ 29 (1) 51.96 AB/CD 0.83 51.74 0.22 0.3185
Afug41
F8 ♀ × M2 ♂ M2 ♂ 27 (3) 48.38 AC/BD 0.89 48.29 0.09 0.3853
F2 ♀ × M4 ♂ F2 ♀ 27 (3) 48.38 AB/CD 0.78 48.03 0.35 0.2776
M4 ♂ 27 (3) 48.38 AB/CD 0.44 45.98 2.39 0.0609
F4 ♀ × M4 ♂ F4 ♀ 29 (1) 51.96
AC/BD
and
AD/BC
0.93 51.93 0.03 0.4259
M4 ♂ 29 (1) 51.96 AD/BC 0.83 51.74 0.22 0.3185
F6 ♀ × M7 ♂ F6 ♀ 28 (2) 50.17 AD/BC 0.86 50.02 0.15 0.3509

Diversity 2022, 14, 745 8 of 17
M7 ♂ 28 (2) 50.17 AB/CD 0.86 50.02 0.15 0.3509
Nacc7 ♀ ×
Nacc5 ♂
Nacc7 ♀ 28 (4) 50.17 AD/BC 0.64 49.21 0.96 0.1631
Nacc5 ♂ 28 (4) 50.17 AD/BC 0.75 49.71 0.46 0.2489
Nacc8 ♀ × Nacc
31♂ Nacc8 ♀ 31 (1) 55.54 AD/BC 0.77 55.13 0.41 0.2603
An20 * F7 ♀ × M8 ♂ M8 ♂ 30 (0) 53.75 AD/BC 0.70 53.03 0.72 0.1984
Anac6784 Nacc19 ♀ ×
Nacc17 ♂ Nacc19 ♀ 30 (2) 53.75
AC/BD
and
AD/BC
0.91 53.68 0.08 0.3912
Anac3133 F7 ♀ × M8 ♂ M8 ♂ 29 (1) 51.96 AB/CD 0.41 49.06 2.90 0.0444 ª
AnacA6 * F5 ♀ × M6 ♂ M6 ♂ 29 (1) 51.96
AB/CD
and
AC/BD
0.72 51.38 0.58 0.2225
AnacB11
F6 ♀ × M7 ♂ M7 ♂ 26 (4) 46.59 AB/CD 0.58 45.31 1.28 0.1290
Nacc19 ♀ ×
Nacc17 ♂ Nacc19 ♀ 26 (6) 46.59 AB/CD 0.81 46.34 0.25 0.3088
AnacB7
F6 ♀ × M7 ♂ F6 ♀ 27 (3) 48.38 AB/CD 0.67 47.57 0.80 0.1849
Nacc19 ♀ ×
Nacc30 ♂ Nacc30 ♂ 32 (0) 57.34 AD/BC 0.94 57.30 0.03 0.4295
AnacE4 * F5 ♀ × M6 ♂ F5 ♀ 28 (2) 50.17
AB/CD
and
AD/BC
0.96 50.16 0.01 0.4622
AoxD161
F4 ♀ × M5 ♂ F4 ♀ 29 (1) 51.96 AB/CD 0.00 40.20 11.76 0.0003
F8 ♀ × M2 ♂ F8 ♀ 30 (0) 53.75 AC/BD 0.10 45.28 8.47 0.0018 ª
Nacc7 ♀ ×
Nacc5 ♂ Nacc7 ♀ 32 0) 57.34 AD/BC 0.00 44.36 12.97 0.0002
AoxD234
*
F2 ♀ × M4 ♂ F2 ♀ 27 (3) 48.38 AD/BC 0.89 48.29 0.09 0.3853
F4 ♀ × M5 ♂ F4 ♀ 28 (2) 50.17 AD/BC 0.86 50.02 0.15 0.3509
M5 ♂ 28 (2) 50.17 AD/BC 0.64 49.21 0.96 0.1631
Nacc28 ♀ ×
Nacc23 ♂
Nacc28 ♀ 23 (1) 41.21 AB/CD 0.78 40.93 0.28 0.2972
Nacc23 ♂ 23 (1) 41.21 AC/BD 0.26 37.29 3.92 0.0239 ª
F6 ♀ × M7 ♂ F6 ♀ 29 (1) 51.96 AC/BD 0.52 50.07 1.89 0.0844
AoxD241
* F7 ♀ × M8 ♂ M8 ♂ 29 (1) 51.96 AC/BD 0.72 51.38 0.58 0.2225
Spl163 F2 ♀ × M4 ♂ M4 ♂ 28 (2) 50.17 AC/BD 0.00 38.82 11.35 0.0004
Spl168
F7 ♀ × M8 ♂ M8 ♂ 26 (4) 46.59 AB/CD 0.00 36.04 10.54 0.0006
F4 ♀ × M5 ♂ F4 ♀ 30 (0) 53.75 AB/CD 0.00 41.59 12.16 0.0002
M5 ♂ 30 (0) 53.75 AB/CD 0.00 41.59 12.16 0.0002
F6 ♀ × M7 ♂ F6 ♀ 29 (1) 51.96 AB/CD 0.00 40.20 11.76 0.0003
M7 ♂ 29 (1) 51.96 AB/CD 0.00 40.20 11.76 0.0003
Nacc19 ♀ ×
Nacc17 ♂
Nacc19 ♀ 23 (9) 41.21 AB/CD 0.00 31.88 9.33 0.0011
Nacc17 ♂ 23 (9) 41.21 AB/CD 0.00 31.88 9.33 0.0011
Nacc7 ♀ ×
Nacc5 ♂
Nacc7 ♀ 32 (0) 57.34 AB/CD 0.00 44.36 12.97 0.0002
Nacc5 ♂ 32 (0) 57.34 AB/CD 0.00 44.36 12.97 0.0002
Nacc8 ♀ ×
Nacc31 ♂
Nacc8 ♀ 29 (3) 51.96 AB/CD 0.00 40.20 11.76 0.0003
Nacc31 ♂ 29 (3) 51.96 AB/CD 0.10 43.86 8.10 0.0022 ª
Results of Likelihood Ratio Test between the null model of tetrasomy (TAU = 1) and the best fitting
intermediate one (TAU estimated). Significant p-values (after Bonferroni correction) that reject the
null hypothesis of tetrasomy are highlighted in grey. ª not significant values after Bonferroni cor-
rection for multiple tests. * Loci for which the segregation inheritance pattern was already explored
in other familiar groups of the same species [12].
Expected allele combinations in tetrasomic and disomic mode of inheritance in tetra-
ploids. (a) Observed allele combinations inherited from a complete heterozygote parent
in autotetraploids in which tetrads are generated during meiosis and random segregation

Diversity 2022, 14, 745 9 of 17
of allele pairs is expected. In brackets, the number of individuals carrying the relative al-
lele combination is reported. (b) Observed allele combinations inherited from a complete
heterozygote parent in allotetraploids in which a preferential pair between homologous
chromosomes occurs. Only four possible gametes are expected. Alleles of the parent for
which the segregation is reported in each table are marked in bold and by a letter used to
indicate the observed combinations. Complete tables for all loci are reported in Appendix
A. (Created with BioRender.com, accessed on 8 August 2022 )
4. Discussion
The segregation pattern observed in the present study at most loci indicates that the
Adriatic sturgeon can be considered predominantly tetraploid with a tetrasomic inher-
itance pattern. The presence of three disomic loci, however, indicates that the functional
diversification process is at different stages in different parts of the genome, with some
regions possibly still octaploid, most tetraploid, and some others in which the degree of
divergence has gone up to a condition of double diploidy. We also observed the presence
of two loci with a marked tendency to disomy with a few unexpected allele combinations,
pointing to a possible imperfect preferential pairing expected at intermediate stages of
functional diploidization [10,11]; this coexistence of different ploidy levels was previously
described in other organisms. In plants, for example, the co-existence of different segre-
gation patterns (e.g., tetrasomic and disomic) with different intermediate degrees of pref-
erential pairing among chromosomes provides evidence of a process of functional reduc-
tion of ploidy which reasonably cannot occur simultaneously throughout the genome, but
which is the result of a progressive differentiation [11,25].
As for the sturgeons, in other species the presence of different degrees of ploidy has
been deduced based on the maximum number of alleles per individual present at the dif-
ferent loci [16] and, in some cases, the Mendelian transmission in the progeny has also
been verified [15,26,27]. However, the selection of the cross/locus combinations to allow
the traceability of every single allele and consequently to distinguish between the different
modes of tetraploid segregation (e.g., disomic, tetrasomic or intermediate) has been con-
ducted to our knowledge only on the Adriatic sturgeon.
The evidence that the level of homology between chromosomes within the Adriatic
sturgeon genome is likely to vary from chromosome to chromosome and that different
parts of the genome may consequently have different degrees of functional ploidy (2 to 8)
should be considered when characterizing the genome of this species and probably of
sturgeons in general. Genome assembly procedures must contemplate the possibility that
different regions are present with a variable number of copies.
Whatever the evolutionary origin and implications of our results, which can be better
investigated only when the genome of this species will be available, the identification of
different inheritance pattern at different markers may have practical consequences for the
conservation of the Adriatic sturgeon. In fact, our findings suggest that before developing
parental allocation methods, a preliminary analysis of the loci used is recommended; this
would ease the reconstruction of individual genealogies of animals kept as captive breed-
ers and the reallocation of any individual recaptured after release. Another interesting
and relatively unexplored aspect of sturgeon ex situ conservation in which a clear
knowledge of the inheritance patterns at different loci could be useful is the monitoring
of the genomic impact of breeding protocols. Captive breeding is usually performed fol-
lowing standardized protocols of hormonal induction of egg and milt release and fertili-
zation is done in an excess of sperms. Then, the resulting viable progeny is released with-
out verifying if the procedure used had some effect on their genomic asset, for example,
by inducing aneuploidies, which are a common phenomenon in some captive sturgeon
stocks [28]. The release in nature of genetically anomalous individuals should be avoided
as the consequences that this can have on the following generations and on their repro-
ductive efficiency is unknown and, in the case of animals with long generation times,
could be revealed after decades. Random screening of familiar groups to verify the correct

Diversity 2022, 14, 745 10 of 17
parents-to-progeny segregation at both disomic and tetrasomic loci could significantly
contribute to reducing the potential impact of genomic anomalies on natural populations.
5. Conclusions
Thanks to the availability of some family groups not previously analyzed, it was pos-
sible to better investigate the patterns of chromosomal segregation in the polyploid Adri-
atic sturgeon. The picture that emerged is that of an extremely dynamic genome in which
it is possible to find the co-existence of regions with different degrees of ploidy, some of
which retain the legacy of ancient duplications and others show a dynamic reduction of
functional ploidy; this pattern is probably shared with other polyploid sturgeons in which
different patterns of segregation have been observed [27], but it is not known whether the
same genomic regions are involved.
Additional studies are required to better characterize the distribution of ploidy across
the genome; this will likely contribute to explaining some specific features of the stur-
geons, such as the ability of species with different degrees of ploidy to hybridize and pro-
duce viable offspring [29]; this extreme genomic plasticity could somehow be linked to a
high degree of genomic redundancy; it would also be very interesting to verify whether
the regions with different segregation patterns are somehow related to the size of the chro-
mosomes, which in sturgeons is known to be very variable, with a small number of large
chromosomes and a high number of micro-chromosomes. However, until complete ge-
nomes of good quality are available, it is difficult to move beyond speculation on these
topics.
Author Contributions: Conceptualization, S.D.P., E.B. and L.C.; methodology, S.D.P., E.B. and L.C.;
investigation, S.D.P. and S.G.B.; writing—original draft preparation, S.D.P. and L.C.; writing—re-
view and editing, E.B., V.H.M.-M. and G.B.; project administration, G.B. and L.C.; funding acquisi-
tion, G.B. and L.C. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Italian PRIN 2017, grant number 201794ZXTL
Institutional Review Board Statement: The animal study protocol was approved by the Institu-
tional Ethics Committee of the University of Padova ( OPBA)—(protocol code 421922 of 14 October
2020).
Informed Consent Statement: Not applicable
Data Availability Statement: Not applicable.
Acknowledgments: We would like to thank the Province of Vicenza and the “Bacino Pesca B (VI)”
for the hospitality in the “Oasi le Sorgenti” Natural Reserve and for the logistical support and the
Storione Ticino aquaculture farm for performing all crosses that allowed this study.
Conflicts of Interest: The authors declare no conflict of interest.
Appendix A

Diversity 2022, 14, 745 11 of 17
.
..
A B C D 304 318 322
293 309 318 330 EFGH
313 318 322 313 338 342 346
AB (2) 293 309 EF (4) 313 338
AC (3) 293 318 EG (1) 313 342
AD (6) 293 330 EH (8) 313 346
BC (2) 309 318 FG (4) 338 342
BD (3) 309 330 FH (8) 338 346
CD (6) 318 330 GH (4) 342 346
313 318 342 212 234 293
E F GH E F GH
293 309 318 322 203 255 259 285
EF (7) 293 309 EF (3) 203 255
EG (7) 293 318 EG (4) 203 259
EH (4) 293 322 EH (4) 203 285
FG (4) 309 318 FG (5) 255 259
FH (4) 309 322 FH (4) 255 285
GH (3) 318 322 GH (7) 259 285
Locus Afug132
Locus Afug132
Observed combinations
F8 ♀ x M2 ♂
Phenotypes
Observed combinations Observed combinations
F5 ♀ x M6 ♂
Phenotypes
F6 ♀ x M7 ♂
Phenotypes
Locus Afug41
F8 ♀ x M2 ♂
Observed combinations
Locus Afug132 Phenotypes
ABCD ABCD
247 255 259 285 247 255 259 285
E F GH E F GH
212 255 293 305 212 255 293 305
AB (4) 247 255 EF (2) 212 255
AC (4) 247 259 EG (5) 212 293
AD (7) 247 285 EH (7) 212 305
BC (3) 255 259 FG (5) 255 293
BD (6) 255 285 FH (6) 255 305
CD (3) 259 285 GH (2) 293 305
ABC D ABC D
212 217 242 259 212 217 242 259
E F GH E F GH
212 255 293 305 212 255 293 305
AB (6) 212 217 EF (6) 212 255
AC (6) 212 242 EG (5) 212 293
AD (4) 212 259 EH (3) 212 305
BC (5) 217 242 FG (5) 255 293
BD (3) 217 259 FH (6) 255 305
CD (5) 242 259 GH (4) 293 305
F4 ♀ x M4 ♂
Observed combinations
Observed combinations
Locus Afug41
F2 ♀ x M4 ♂
Phenotypes
Phenotypes
F4 ♀ x M4 ♂
Observed combi nations
Observed combi nations
F2 ♀ x M4 ♂
Locus Afug41
Locus Afug41 Locus Afug41
Phenotypes
Phenotypes

Diversity 2022, 14, 745 12 of 17
.
..
A B C D 304 318 322
293 309 318 330 EFGH
313 318 322 313 338 342 346
AB (2) 293 309 EF (4) 313 338
AC (3) 293 318 EG (1) 313 342
AD (6) 293 330 EH (8) 313 346
BC (2) 309 318 FG (4) 338 342
BD (3) 309 330 FH (8) 338 346
CD (6) 318 330 GH (4) 342 346
313 318 342 212 234 293
E F GH E F GH
293 309 318 322 203 255 259 285
EF (7) 293 309 EF (3) 203 255
EG (7) 293 318 EG (4) 203 259
EH (4) 293 322 EH (4) 203 285
FG (4) 309 318 FG (5) 255 259
FH (4) 309 322 FH (4) 255 285
GH (3) 318 322 GH (7) 259 285
Locus Afug132
Locus Afug132
Observed combinations
F8 ♀ x M2 ♂
Phenotypes
Observed combinations Observed combinations
F5 ♀ x M6 ♂
Phenotypes
F6 ♀ x M7 ♂
Phenotypes
Locus Afug41
F8 ♀ x M2 ♂
Observed combinations
Locus Afug132 Phenotypes
ABCD ABCD
247 255 259 285 247 255 259 285
E F GH E F GH
212 255 293 305 212 255 293 305
AB (4) 247 255 EF (2) 212 255
AC (4) 247 259 EG (5) 212 293
AD (7) 247 285 EH (7) 212 305
BC (3) 255 259 FG (5) 255 293
BD (6) 255 285 FH (6) 255 305
CD (3) 259 285 GH (2) 293 305
ABC D ABC D
212 217 242 259 212 217 242 259
E F GH E F GH
212 255 293 305 212 255 293 305
AB (6) 212 217 EF (6) 212 255
AC (6) 212 242 EG (5) 212 293
AD (4) 212 259 EH (3) 212 305
BC (5) 217 242 FG (5) 255 293
BD (3) 217 259 FH (6) 255 305
CD (5) 242 259 GH (4) 293 305
F4 ♀ x M4 ♂
Observed combinations
Observed combinations
Locus Afug41
F2 ♀ x M4 ♂
Phenotypes
Phenotypes
F4 ♀ x M4 ♂
Observed combi nations
Observed combi nations
F2 ♀ x M4 ♂
Locus Afug41
Locus Afug41 Locus Afug41
Phenotypes
Phenotypes

Diversity 2022, 14, 745 13 of 17
..
..
..
307 313 148 150
EFGH EFG H
293 297 301 307 138 144 148 162
EF (1) 293 297 EF (2) 138 144
EG (7) 293 301 EG (6) 138 148
EH (0) EH (3) 138
FG (15) 297 301 FG (4) 144 148
FH (0) FH (8) 144 162
GH (6) 301 307 GH (3) 148 162
Locus A6
Observed combinations
F5 ♀ x M6 ♂ F6 ♀ x M7 ♂
PhenotypesPhenotypes
Observed combinations
Locus B11
ABCD ABCD
132 136 138 144 166 172 174 176
132 150 162 154 156 164
AB (4) 132 136 AB (3) 166 172
AC (7) 132 138 AC (8) 166 174
AD (3) 132 144 AD (6) 166 176
BC (5) 136 138 BC (5) 172 174
BD (4) 136 144 BD (2) 172 176
CD (3) 138 144 CD (3) 174 176
Locus B11
Observed combinations
Observed combinations
Locus B7
Nacc19♀ x
Nacc17♂ F6 ♀ x M7 ♂
Phenotypes Phenotypes
156 166 174 A B C D
EFGH 332 340 346 354
154 170 174 176 336 346
EF (8) 154 170 AB (3) 332 340
EG (8) 154 174 AC (5) 332 346
EH (4) 154 176 AD (3) 332 354
FG (6) 170 174 BC (6) 340
FH (3) 170 176 BD (5) 340 354
GH (3) 174 176 CD (6) 346 354
ABC D A BCD
123 127 131 139 123 131 135 139
131 135 155 127 131
AB (0) AB (7) 123 131
AC (10) 123 131 AC (1) 123 135
AD (7) 123 139 AD (6) 123 139
BC (6) 127 131 BC (5) 131 135
BD (6) 127 139 BD (0)
CD (0) CD (11) 135 139
F4 ♀ x M5 ♂
F5 ♀ x M6 ♂
Nacc19♀ x
Nacc30♂
Locus E4
Locus B7
Observed combinations
Observed combinationsObserved combinations
Locus Aox161
Locus Aox161
Observed combinations
Phenotypes
F8 ♀ x M2 ♂
Phenotypes
PhenotypesPhenotypes

Diversity 2022, 14, 745 14 of 17
...
..
...
..
ABC D AB CD
123 127 131 143 219 223 243 255
131 135 139 227 243
AB (24) 123 127 AB (8) 219 223
AC (6) 123 131 AC (5) 219 243
AD (0) AD (4) 219 255
BC (0) BC (4) 223 243
BD (7) 127 143 BD (4) 223 255
CD (5) 131 143 CD (2) 243 255
Locus Aox234
Observed combinations
F2 ♀ x M4 ♂Nacc7♀ x Nacc5♂
Phenotypes
Observed combinations
Locus AoxD161 Phenotypes
A BCD A BCD
227 243 247 251 227 243 247 251
EF GH EF GH
235 239 255 259 235 239 255 259
AB (6) 227 243 EF (8) 235 239
AC (4) 227 247 EG (4) 235 255
AD (6) 227 251 EH (4) 235 259
BC (2) 243 247 FG (2) 239 255
BD ( 7) 243 251 FH (4) 239 259
CD (3) 247 251 GH (6) 255 259
Locus Aox234
Observed combinations
Observed combinations
Locus Aox234
F4 ♀ x M5 ♂ F4 ♀ x M5 ♂
Phenotypes Phenotypes
ABCD ABCD
219 243 247 263 219 243 247 263
EF GH EF GH
239 243 251 255 239 243 251 255
AB (4) 219 243 EF (6) 239 243
AC (7) 219 247 EG (2) 239 251
AD (3) 219 263 EH (6) 239 255
BC ( 4) 243 247 FG (5) 243 251
BD (3) 243 263 FH (0)
CD (2) 247 263 GH (4) 251 255
Phenotypes
Nacc28♀ x Nacc23♂
Locus Aox234
Observed combinations
Observed combinations
Locus Aox234
Nacc28♀ x Nacc23♂
Phenotypes
A B C D 168 176 180
227 243 247 255 EFGH
219 239 247 164 172 176 184
AB (3) 227 243 EF (2) 164 172
AC (1) 227 247 EG (2) 164 176
AD (8) 227 255 EH (5) 164 184
BC (5) 243 247 FG (9) 172 176
BD (4) 243 255 FH (5) 172 184
CD (8) 255 GH (6) 176 184
Locus Aox234 Locus AoxD241
Observed combinations
Observed combinations
Phenotypes
F6 ♀ x M7 ♂ F7 ♀ x M8 ♂
Phenotypes
207 215 220 232 240 294
EFGH EFGH
166 215 224 229 218 236 271 273
EF (11 ) 166 215 EF (0)
EG (0) EG (5) 218 271
EH (5) 166 229 EH (6) 218 273
FG (7) 215 224 FG (7) 236 271
FH (0) FH (8) 236 273
GH (5) 224 229 GH (0)
F2 ♀ x M4 ♂
Phenotypes Locus Spl168
Observed combinations
Observed combinations
Locus Spl163
F7 ♀ x M8 ♂
Phenotypes

Diversity 2022, 14, 745 15 of 17
.
..
..
.
A BCD A BCD
209 232 257 273 209 232 257 273
EF GH EF GH
218 232 279 282 218 232 279 282
AB (0) EF (0)
AC (10) 209 257 EG (7) 218 279
AD (6) 209 273 EH (2) 218 282
BC ( 7) 232 257 FG (13) 232 279
BD (7) 232 273 FH (8) 232 282
CD (0) GH (0)
F4 ♀ x M5 ♂ F4 ♀ x M5 ♂
PhenotypesPhenotypes
Observed combinations
Observed combinations
Locus Spl168
Locus Spl168
AB CD AB C D
227 236 271 286 227 236 271 286
EFGH EFGH
240 245 264 269 240 245 264 269
AB (0) EF (0)
AC (6) 227 271 EG (3) 240 264
AD (4) 227 286 EH (15) 240 269
BC (9) 236 271 FG (6) 245 264
BD ( 10) 236 286 FH (5) 245 269
CD (0) GH (0)
Locus Spl168
Locus Spl168
Observed combinations
Observed combinations
F6 ♀ x M7 ♂
Phenotypes
F6 ♀ x M7 ♂
Phenotypes
ABCD ABCD
218 227 269 294 218 227 269 294
EF G H EF G H
227 240 286 314 227 240 286 314
AB (0) EF (0)
AC (4) 218 269 EG (4) 227 286
AD (7) 218 294 EH (5) 227 314
BC (3) 227 269 FG (8) 240 286
BD (9) 227 294 FH (6) 240 314
CD (0) GH (0)
Observed combinations
Locus Spl168
Observed combinations
Locus Spl168
Nacc19♀ x Nacc17♂
PhenotypesPhenotypes
Nacc19♀ x Nacc17♂
Phenotypes
ABC D ABC D
232 245 264 294 232 245 264 294
EF GH EF GH
214 240 273 279 214 240 273 279
AB (0) EF (0)
AC (12) 232 264 EG (8) 214 273
AD (9) 232 294 EH (7) 214 279
BC (5) 245 264 FG (7) 240 273
BD ( 6) 245 294 FH (10) 240 279
CD (0) GH (0)
Nacc7♀ x
Nacc5♂
PhenotypesLocus Spl168
Locus Spl168
Observed combinations
Observed combinations
Nacc7♀ x
Nacc5♂

Diversity 2022, 14, 745 16 of 17
..
Figure A1. Total segregation results. Single-locus microsatellite inheritance for each studied stur-
geon family. Microsatellite alleles of parents are reported as sizes in bp. For families in which both
parents are informative, two different schemes are shown. The 4 alleles of the informative parents
are highlighted in bold and marked with capital letters A, B, C, D for females and E, F, G, H for
males, also used to label allele combinations observed in the progeny. The number of F1 in which
each combination has been observed is reported in brackets. Missing combinations are marked in
grey.
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ABCD ABCD
209 236 271 279 209 236 271 279
EFGH EFGH
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AB (0) EF (1) 218 249
AC (9) 209 271 EG (8) 218 273
AD (7) 209 279 EH (4) 218 279
BC (6) 236 271 FG (11) 249 273
BD (7) 236 279 FH (5) 249 279
CD (0) GH (0)
Nacc8♀ x Nacc 31♂Nacc8♀ x Nacc 31♂
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