MULTIGENERATIONAL OUTCOMES OF OUTCROSSING IN NATURAL
POPULATIONS
The theoretical possibilities reviewed above highlight the potentially
complex set of fitness outcomes from outcrossing due to natural
immigration across spatially structured populations. We therefore
conducted a literature review, aiming to evaluate the degree to which
key effects have actually been estimated in wild populations where
immigrants naturally outbreed with resident individuals, and thereby
synthesise the overall body of empirical knowledge. We used search terms
on Web of Science aimed to find ecological studies that estimate the
fitness of the offspring of natural immigrants, including studies that
do not specifically interpret results within the heterosis framework, as
well as more traditional heterosis-related search terms (Fig. 5). Due to
the high number of results initially found, we filtered for studies
within the Web of Science category of “ecology or evolutionary
biology”, aiming to exclude studies on domesticated or agricultural
lines, resulting in 12,862 studies after duplicate removal. We validated
our search by comparing the results to a list of previously known
studies by our research group, and triaged the results based on the
series of criteria presented in Table 1. The definition of criteria and
the triage were done by DG in consultation with HJ and JMR.
We found 111 studies (0.86% of 12,862) on natural populations with at
least one estimate of fitness or a fitness-related trait for both within
and between population crosses for at least one generation (criteria
1-5; Supplementary Table S1). These studies exclusively focused on
animals or plants (Fig. 6), encompassing 89 different species. Focal
fitness metrics and traits varied across studies, but commonly included:
germination, biomass, number of flowers, fruits and seeds produced and
seed mass in plants; and number and size of offspring produced (e.g.
clutch and egg size) and developmental time in animals. For both plants
and animals, survival across different life stages and metrics of
cumulative fitness were also common.
Over a third of the 111 studies (39.6%, N=44) presented crosses between
populations for which the degree of connectivity was unclear (Fig. 7),
whether due to a lack of knowledge regarding dispersal in the focal
populations, lack of mention of it within the paper, or because the
information presented was not sufficient for a categorization
considering our specified criteria (Table 1, criteria 6). Another large
proportion of studies (45%, N=50) included heterosis estimates for
hybrid offspring resulting from introduction (N=1) or artificial crosses
(N=49) between populations further than the distance of natural
dispersal, such as crosses between populations separated by several
hundreds of kilometers, and even populations from different continents.
More than half of these studies (N=29) only estimated heterosis at this
level of connectivity, while the others (N=21) combined estimates across
different levels of connectivity (category “both” in Fig. 7). Among
the remaining 17 studies (15%), which we could classify as only
studying populations that are, or possibly are, interconnected by
natural dispersal, only three reported fitness estimates for hybrid
offspring resulting from interbreeding due to natural immigration (Fig.
7).
In two of the three studies conducted under the context of natural
dispersal (Marr et al. 2002, Martinig et al. 2020), estimates were for
the hybrid offspring between residents and natural immigrants, whereby
long-term fitness and parentage data (18-29 years) allowed the
categorization of individuals into resident or immigrant, and their
offspring into the relevant filial generations. While Marr and
collaborators (2002) explicitly aimed to estimate the fitness
consequences of outcrossing following natural dispersal into an island
population of song sparrows (Melospiza melodia ), Martinig and
collaborators (2020) aimed to investigate sex-specific fitness
consequences of dispersal in the North American red squirrels
(Tamiasciurus hudsonicus ).
In the song sparrows, male immigrants showed lower reproductive
performance than residents, but no difference in survival. Among
comparisons of reproductive performance and survival including hybrid
filial generations, no difference was found for hybrid
F1s versus average immigrant-resident, hybrid
F1s versus F2s, and hybrid
F2s versus average F1-immigrant-resident
(Marr et al. 2002). However, F1s produced between 27.1%
(females) to 30.2% (males) more offspring than the average between
immigrant and residents, and about 2 (females) to 3 times (males) more
offspring than F2s. F2 males also showed
decreased survival in comparison to F1s in both juvenile
(46%) and adult (35.1%) stages. These heterotic effects are likely
biologically significant, and the lack of strong statistical evidence
for these differences possibly resulted from low power due to a small
number of immigrants (14 females and 4 males vs. >100
residents). In turn, male immigrants of North American red squirrels had
longer lifespans and produced more offspring than residents, while
female immigrants had reduced lifetime reproductive success (Martinig et
al. 2020). In addition, researchers found that both immigrant males and
females produced daughters with lower lifetime reproductive success than
resident males and females, respectively. However, no further
comparisons or specifications of filial generations were presented, nor
a distinction was made between offspring of immigrant-immigrant versus
immigrant-resident crosses.
The third study (Bull & Sunnucks 2014) investigated the fitness of
hybrid offspring along a contact zone between two races of a velvet worm
(Euperipatoides rowelli ), aiming to investigate the maintenance
of morphological differentiation between the races. Using body
coloration patterns and haplotypes to classify individuals and embryos
as either of the races or as hybrids, researchers found no evidence for
a difference in embryo-to-adult survival across categories. However,
hybrids represented a single category for which genetic admixture
proportions deviated 15% in either direction from the pure parental
races and, therefore, likely included different filial generations.
The remaining studies classified as using populations that were or
likely to be connected by natural dispersal (N=14) conducted
experimental crosses to obtain hybrid offspring. Most of these studies
estimated fitness under artificial or semi-natural conditions (Fig. 7),
with ten studies (71.4% of 14) investigating fitness within a single
environmental condition, and only five estimating fitness for at least
one component under at least one of the native parental environments.
None of the studies using experimental crosses explicitly considered sex
differences in F1 heterosis, likely due to focusing on
hermaphroditic [plant] species or pre-sexual maturation life stages
in animals. Moreover, only five of the 14 studies (35.7%) considered
filial generations beyond the F1, including estimates
for F2 (N=2), F3 (N=1) or backcrosses
(N=3). These observations do not appreciably change when considering all
of the 111 studies, with 70.3% (N=78) of the studies including only
estimates for the F1 offspring, 70.3% including
estimates for a single environmental condition, and only one study
(Matsubayashi et al. 2011) that quantified male- and female-specific
traits.
Among the studies using experimental crosses that included populations
that are or likely are connected by natural dispersal (i.e. categories
“Connected” and “Both” on Fig. 7), a mixture of evidence for
positive, negative, or no heterosis was found. Heterosis often varied
within studies, among traits or pairwise population combinations, and
even among replicate crosses of the same populations. In some cases,
such differences resulted from rearing environment (Ostevik et al. 2016)
or maternal population of origin (Barnard-Kubow et al. 2016,
Barnard-Kubow & Galloway 2017), demonstrating that outcomes may depend
on environmental and on maternal effects, cytoplasmatic-nuclear genome
interactions (Burton et al. 2006), or sex-chromosomal effects (Saavedra
& Amat 2005). Some studies also found a relationship between heterosis
and the genetic (Barker et al. 2019, Barnard-Kubow et al. 2016,
Barnard-Kubow & Galloway 2017), geographical (Barnard-Kubow et al.
2016) or environmental distance (Pickup et al. 2013) between parental
populations, or the size and the genetic diversity within populations
(Pickup et al. 2013, Willi et al. 2007). Genetic or geographical
distance (Barnard-Kubow et al. 2016, Barnard-Kubow & Galloway 2017,
Willi & Van Buskirk 2005) and genetic diversity (Barker et al. 2019,
Pickup et al. 2013, Willi & Van Buskirk 2005, Willi et al. 2007),
however, were also reported to not affect heterosis in other studies or
in different traits, environments, or filial generations within the same
study. As an interesting example, fitness of hybrid offspring of the
herb Campanulastrum americanum showed no relationship with
genetic distance estimated from nuclear markers, but decreased with the
genetic distance estimated from the predominantly maternally inherited
chloroplast markers (Barnard-Kubow et al. 2016, Barnard-Kubow &
Galloway 2017; but see Barnard-Kubow et al. 2017). Even further,
maternal population of origin reversed the direction of heterosis within
both F1 and backcrosses of reciprocal crosses for some
traits and population combinations (Barnard-Kubow et al. 2016).
Therefore, cytoplasmatic-nuclear interactions may not just affect
heterosis levels per se, but also the correlation between heterosis and
genetic distance.
Unfortunately, our review also revealed that explicitly quantifying and
contrasting conditions under which heterosis is positive, negative, or
absent across studies is constrained by forms of analyses and data
reporting. Papers commonly presented a single statistical estimate for
crosses between several populations, sometimes of different connectivity
levels. Moreover, criteria for determination of heterosis varied
enormously across studies, ranging from statistical comparisons between
mean values of hybrid F1 and mean parental values, to
more elaborate calculations involving the average offspring values of
within and between population crosses (Supplementary Table S2). Finally,
these studies also often implement methodological priorities that may
introduce errors or biases within the context of our research agenda.
For example, many study designs used a substantial number of offspring
(e.g. >20) from a small number of parents (e.g.
<5 parent pairs), rather than using offspring generated from
more parental combinations. Also common was the use of multiple sires or
pollen mixtures (sometimes from different source populations) for
fertilization, with the aim of reducing the probability of unsuccessful
crosses. Further, unsuccessful crosses were sometimes replaced, to
ensure estimation of fruit or flower productivity in the offspring.
Although these tactics are justified under alternative research
contexts, key information regarding differences across families,
pairwise population combinations, degrees of connectivity, or
environmental characteristics is lost. Conclusions are also muddied by
averaging of positive and negative outcomes within individual
generations or pairwise population combinations. Furthermore,
eliminating unsuccessful crosses from the data, or removing or
confounding individual level variation via use of single or mixed sires,
restricts proper variance and error estimations in the statistical
analyses, and prevent documentation of cases in which outcrossing does
not result in viable offspring. Therefore, estimates and conclusions
about heterotic effects from these studies may not provide adequate
evidence for heterotic effects within the context of eco-evolutionary
consequences of dispersal in wild populations.