H . Zhou J. Exp. Mar. Biol. Ecol. 256 2001 99 –121
105
and Warwick, 1994. Changes in nematode community structure were examined using non-parametric multivariate techniques contained in the PRIMER Plymouth Routines in
Multivariate Ecological Research package. MDS non-metric Multidimensional Scaling Ordination was based on the Bray–Curtis similarity of either single square root
transformed or untransformed, nematode species abundance data. Untransformed data weight more contributions on common rather than rare species compared with the single
square root transformation Clarke and Warwick, 1994. The ANOSIM Analysis of Similarity technique was used to test treatment and time effects on the nematode
community succession process. A SIMPER Similarity Percentages program was then employed to identify those species contributing to differences between field and
experimental samples and between treatments observed in the MDS and ANOSIM analysis Clarke and Warwick, 1994.
For both the univariate and multivariate analyses, a significance level of P 0.05 was used as the rejection value. Field control samples F were also analyzed with
experimental samples C, L, M and H whenever appropriate.
3. Results
3.1. Major taxa Sixteen taxa were recorded during this experiment. Nematodes were the most
dominant group in general, averaging 88 of the total meiofauna over all sampling dates in the field control samples, and 31, 35, 57 and 78 in the control, low, medium and
high dose tubes, respectively. Copepods nauplii in parentheses accounted for only 2 0 of the total meiofauna in the ambient sediment, but 46 6, 42 6, 28
3 and 13 1 in the control, low, medium and high dose tubes, respectively. Polychaetes had a relatively constant average dominance around 8 over all sampling
dates in the field controls and all levels of treatment. Other groups, including oligochaetes, ostracods, halacaroids, insect larvae, kinorynchs, turbellarians, amphipods,
bivalves, isopods, pycnogonids and tanaids, were found in low numbers only and were, thus, not focused upon in this study.
Initial meiofaunal colonization occurred 1 day post-placement, but at a slower rate in the detritus samples than the controls Table 2; Fig. 3. Copepods colonized faster than
nematodes and polychaetes, but seemed more sensitive to the newly-added detritus. That is, copepod numbers in the controls 14618 ind. per core were close to that of field
control samples 18623 ind. per core after only 1 day, while samples with detritus additions had no copepods. Compared with the numbers in the field control samples
6096294, nematodes initially colonized the experimental sediments in low numbers 564 in controls and 161 in detritus samples.
Meiofaunal densities generally increased during the course of the experiment, regardless of treatment effects Fig. 3A–D. This was not unexpected for a colonization
process. In the organic-free control sediment, nematode and polychaete numbers increased with time, but at a lower rate than for the copepods Table 2; Fig. 3. After 60
days, nematodes achieved only 20 and polychaetes 60 of their background numbers.
106 H
. Zhou J. Exp. Mar. Biol. Ecol. 256 2001 99 –121 Table 2
2
Mean numbers 6S.D., n 5 4 per core 6.15 cm of major meiofaunal taxa and a bacterial nematode Diplolaimella sp. recorded from each treatment on each sampling date during the experimental period. F, field
control; C, control; L, low dose; M, medium dose; H, high dose Time
Treat- Nematoda
Diplolaimella Copepoda
Polychaeta Total
ment sp.
meiofauna 1 Day
F 6096294
18623 69629
7126219 C
564 14618
162 22622
L 161
160 M
161 161
H 161
161 10 Days
F 9396144
1765 105634
10846173 C
68638 109652
1464 241668
L 90631
28618 56626
1768 176662
M 75666
32649 21617
663 106684
H 40620
869 1668
664 64631
30 Days F
11806275 264
1669 60649
12926322 C
104622 261
146657 31627
340694 L
4076250 2806186
150663 55634
6496364 M
3436104 159655
83682 65639
4996209 H
3606220 2186168
91639 70629
5276216 60 Days
F 19176504
364 1365
116626 20676479
C 4006117
46639 91663
70653 6036104
L 13476498
7616352 145687
33629 15546622
M 379762206
318662060 1246117
36622 397862271
H 730165096
582465269 6236603
74630 815565676
Copepod numbers 109652 were, however, as much as 63 the background levels 1765 after 10 days and reached the highest value 146657 30 days post-placement,
declining to 91663 on the 60th day. In contrast, nematode numbers increased much faster in the detritus samples than in the controls. By the end of the experiment 60 days
post-placement, numbers reached the highest value of only 0.23 the background level in the control, but 0.73, 23 and 43 background levels for the low, medium and high
dose additions, respectively.
Two-way ANOVA revealed that interactions of the main effects, i.e. time and treatment, were significant for densities of total meiofauna and all its major taxa Table
3. This suggested that treatment effects on meiofauna densities varied over time. One-way ANOVA Table 4 carried out for treatment effects on meiofaunal densities at
a specific stage of the experiment showed that they were significant for total meiofauna 1 and 60 days, nematodes 30 and 60 days, copepods 1 and 10 days and polychaetes
10 days. Responses of meiofauna to the leaf litter addition indicated two different treatment effects on meiofaunal colonization: a negative effect was identified as faunal
densities decreased with increased levels of leaf litter additions and vice versa. Negative effects of leaf litter addition showed up 1 day post-placement for the total meiofauna
C.L5M5H, copepods from days 1 C.L5M5H to 10 C.L.M5H and polychaetes after 10 days C5L.M5H Table 4; Fig. 3. Positive responses of the
H . Zhou J. Exp. Mar. Biol. Ecol. 256 2001 99 –121
107
Fig. 3. A–E Densities ind. per core of major meiofaunal groups and a bacterial feeding nematode Diplolaimella sp.; F nematode species number. Error bar represents mean61 S.D. n 5 4; C, control; L, low
dose; M, medium dose; H, high dose; F, field control. All data are lnx 1 1 transformed.
108 H
. Zhou J. Exp. Mar. Biol. Ecol. 256 2001 99 –121 Table 3
Results from two-way ANOVA for the time and treatment effects on numbers ind. per core of major meiofaunal taxa and a bacterial feeding nematode Diplolaimella sp. and nematode species numbers. Field
controls are not included in these analyses. Day 1 is not included as few meiofauna colonized each tube. A Bonferroni multiple comparison for nematode species numbers is shown in parentheses
Source df
P of
Total Nematoda
Copepoda Polychaeta
Diplolaimella Nematode
variation meiofauna
sp. species
Time days 2
,0.001 ,0.001
,0.001 ,0.001
,0.001 ,0.001
10530,60 Treatment
3 0.169
0.053 0.055
0.631 ,0.001
0.030 C.L5M5H
Time 6
,0.001 ,0.001
0.002 0.031
0.031 0.842
3Treatment Total
48
meiofauna to leaf litter additions became significant at the later stages of the experiment, i.e. total meiofauna on the 60th day H5M.L.C and nematodes on the 30th
H5M5L.C and 60th day H5M.L.C Table 4; Fig. 3. No significantly positive effects for copepods and polychaetes could be detected by the end of the
experiment.
The rapid increase in nematode numbers in the detritus samples was due to one species, i.e. Diplolaimella sp., which was otherwise rare in controls and ambient
mangrove sediments. It reached an average density of 5824 ind. per core in the high dose addition samples on the 60th day Table 2. An interaction of time and treatment
effects on the numbers of Diplolaimella sp. was also revealed by two-way ANOVA Table 3. The colonization of Diplolaimella sp. started 10 days post-placement and
showed significant treatment effects up to the end of the experiment. After 60 days, a positive treatment gradient was clear, i.e. H5M.L4C Table 4; Fig. 3.
Table 4 Significance levels from one-way ANOVA for the treatment effects on densities of major meiofaunal taxa and
a bacterial feeding nematode, Diplolaimella sp., at each specific stage of the experiment. Field controls are not included in these analyses
Days post- Total
Nematoda Copepoda
Polychaeta Diplolaimella
placement meiofauna
sp.
a b
a
1 0.020
– 0.002
– –
10 0.110
0.298 0.002
0.023 0.006
30 0.590
0.049 0.251
0.415 ,0.001
a a
60 0.008
0.007 0.089
0.173 ,0.001
a
Kruskal–Wallis test instead of ANOVA.
b
–, no test performed as few animals colonized.
H . Zhou J. Exp. Mar. Biol. Ecol. 256 2001 99 –121
109
3.2. Nematode community 3.2.1. Diversity
Two-way ANOVA Table 3 indicated significant time and treatment effects of leaf litter addition on nematode species numbers during the experimental period. The time
and treatment interaction was not significant, however. The Bonferroni test Table 3 showed that nematode species numbers of 10 and 30 days post-placement were
significantly less than after 60 days and leaf litter addition significantly reduced them also C.L5M5H. K-dominance curves Fig. 4 plotted for different stages of the
experiment revealed that: first, the presence of leaf litter reduced nematode species diversity; second, dose effects changed over the experimental time, so that by the end of
the experiment, a negative dose effect was observed, i.e. C.L.M5H; and third, after
Fig. 4. K-dominance curves of nematode species assemblage calculated by averaging replicates for each treatment level after 10, 30 and 60 days, respectively. F, field control; C, control; L, low dose; M, medium
dose; H, high dose.
110 H
. Zhou J. Exp. Mar. Biol. Ecol. 256 2001 99 –121
30 days, diversity of the ambient nematode community F was higher than that of the detritus samples L, M and H but lower than the controls C.
3.2.2. Community structure Nematode community structure in the experimental sediment changed over the
colonization process while the community structure in the colonizing pool was rather stable, as revealed by MDS Figs. 5 and 6. Detritus addition affected community
colonization until the end of the experiment and there were always large differences between field and experimental samples during the whole study period Fig. 6.
A formal test of experimental effects on the nematode community structure using two-way crossed ANOSIM showed a highly significant time effect for both the global
and pair-wise tests between different times Table 5. A treatment effect was highly significant only between controls and detritus samples. One-way ANOSIM further
revealed that treatment effects were significant between controls and the detritus samples after 10 days Table 6. Dose effects were not so obvious and could only be detected on
the 60th day between L and M Table 6 and L and H Tables 5 and 6 dose regimes.
3.2.3. Species composition and trophic structure Most of the species in the experimental sediments are common in the natural
Fig. 5. MDS ordination based on single square root transformed species abundance data using Bray–Curtis similarity, showing changes in nematode community structure over the experimental period. Each sample is
identified by a treatment code C, control; L, low dose; M, medium dose; H, high dose and a subscript representing experimental time 051 day, 1510 days, 3530 days, 6560 days. Field controls F not
included.
H . Zhou J. Exp. Mar. Biol. Ecol. 256 2001 99 –121
111
Fig. 6. MDS ordinations of nematode community structure over the experimental period based on untrans- formed A and single square root transformed B abundance data using Bray–Curtis similarity. Field controls
F are also included and replicates pooled over each sampling occasion for simplification. Abbreviations as used in Fig. 5.
environment of the study site, except for the two extreme cases, i.e. Diplolaimella sp., which was dominant in the detritus samples from day 10 onwards but rare in controls
and in the ambient mangrove sediment, and Parastomanema sp., a mouthless nematode which, conversely, was abundant in the natural environment but rarely colonized into the
experimental sediments. The top three species contributing to the identified differences in community structure between field and experimental samples and between different
levels of treatment at a specific stage of the experiment were indicated by SIMPER analysis and listed in Tables 7 and 8. Experimental samples differed from field controls
due to Parastomanema sp. 10, 30 and 60 days, Sabateria praedatrix De Man 10, 30 and 60 days, Metalinhomoeus biformis Juario 30 days and Chromaspirina sp. 10
days, which were more abundant in the natural environment. The major contributor to the differences between controls and detritus samples was Diplolaimella sp. 10, 30 and
Table 5 Results from two-way crossed ANOSIM global and pair-wise tests using Bray–Curtis similarity, showing the
overall time and treatment effects on nematode community structure during the colonization experiment. R
u
and R : R values based on untransformed and single square root transformed abundance data, respectively. C,
t
control; L, low dose; M, medium dose; H, high dose Time days
Treatment Global: R 5 0.744 P , 0.001
Global: R 5 0.429 P , 0.001
u u
R 5 0.717 P , 0.001 R 50.400 P , 0.001
t t
Pair-wise R P
R P Pair-wise
R P R P
u t
u t
1 and 10 0.700 ,0.001
0.676 ,0.001 C and L
0.622 ,0.001 0.562 ,0.001
1 and 30 0.802 ,0.001
0.784 ,0.001 C and M
0.712 ,0.001 0.656 0.001
1 and 60 0.786 ,0.001
0.786 ,0.001 C and H
0.806 ,0.001 0.743 ,0.001
10 and 30 0.604 ,0.001
0.536 ,0.001 L and M
0.134 0.078 0.050 0.298
10 and 60 0.977 ,0.001
0.943 ,0.001 L and H
0.203 0.028 0.140 0.099
30 and 60 0.784 ,0.001
0.792 ,0.001 M and H
0.008 0.431 0.029 0.380
112
H .
Zhou J
. Exp
. Mar
. Biol
. Ecol
. 256
2001 99
– 121
Table 6 Results from one-way ANOSIM global and pair-wise tests using Bray–Curtis similarity, showing the treatment effects on nematode community structure at a specific
experimental stage. Abbreviations as used in Table 5
10 Days 30 Days
60 Days Global:
R 5 0.401 P 5 0.002 R 5 0.451 P 5 0.003
R 5 0.591 P 5 0.001
u u
u
R 5 0.357 P 5 0.004 R 5 0.429 P 5 0.002
R 5 0.569 P , 0.001
t t
t
Pair-wise R P
R P R P
R P R P
R P
u t
u t
u t
C and L 0.740 0.029
0.688 0.029 0.698 0.029
0.677 0.029 0.917 0.029
0.656 0.029 C and M
0.615 0.029 0.573 0.029
1.000 0.029 0.938 0.029
1.000 0.029 0.948 0.029
C and H 0.781 0.029
0.760 0.029 1.000 0.029
0.813 0.029 0.938 0.029
0.896 0.029 L and M
20.146 0.829 20.292 0.971
0.125 0.200 0.219 0.171
0.427 0.029 0.208 0.057
L and H 0.271 0.057
0.302 0.086 20.010 0.571
20.177 0.829 0.438 0.029
0.365 0.029 M and H
0.167 0.171 0.042 0.429
20.115 0.743 20.104 0.686
0.010 0.429 0.187 0.143
H . Zhou J. Exp. Mar. Biol. Ecol. 256 2001 99 –121
113 Table 7
SIMPER analysis of the nematode community identifying the top three species making the biggest percentage contributions to Bray–Curtis dissimilarities based on single square root transformed abundance data
between field controls and experimental samples at a specific stage of the experiment. F, field control; C, control; L, low dose; M, medium dose; H, high dose
Samples 10 Days
30 Days 60 Days
compared Species
Species Species
F and C Parastomanema sp.
15.5 Parastomanema sp.
15.2 Parastomanema sp.
11.9
a
Sabateria praedatrix 6.7
Sabateria praedatrix 7.5
Diplolaimella sp. 6.5
Chromaspirina sp. 6.5
Metalinhomoeus biformis 5.1
Sabateria praedatrix 5.1
a
F and L Parastomanema sp.
13.6 Parastomanema sp.
13.3 Diplolaimella sp.
13.0
a
Sabateria praedatrix 5.9
Diplolaimella sp. 9.4
Parastomanema sp. 10.9
Chromaspirina sp. 5.8
Sabateria praedatrix 6.3
Sabateria praedatrix 4.7
a
F and M Parastomanema sp.
13.9 Parastomanema sp.
13.0 Diplolaimella sp.
24.6
a
Chromaspirina sp. 6.3
Diplolaimella sp. 7.4
Parastomanema sp. 9.0
Sabateria praedatrix 6.1
Sabateria praedatrix 6.2
Sabateria praedatrix 3.9
a
F and H Parastomanema sp.
12.2 Parastomanema sp.
12.7 Diplolaimella sp.
27.3
a
Chromaspirina sp. 5.5
Diplolaimella sp. 8.4
Parastomanema sp. 7.9
a
Sabateria praedatrix 4.9
Sabateria praedatrix 6.2
Anoplostoma viviparum 4.1
a
Average density of the species is higher in the latter sample.
60 days. Other important species included Diplolaimelloides sp. 10, 30 and 60 days, Theristus sp. 10 and 30 days, Haliplectus wheeleri Coles 30 days, Megadesmolaimus
sp. 60 days and Anoplostoma viviparum Bastian 60 days in the detritus samples and an unidentified species 10 days in the controls. Some epi-growth feeders Type 2A
also showed a positive response to the detritus 60 days post-placement, i.e. Desmodora cazca Gerlach L.C, Dichromadora sp. H.C, Chromaspirina sp. H.L and
Paracanthochus sp. H.M.
Nematode trophic composition, as illustrated in Fig. 6, was based on Wieser’s 1953 four trophic group scheme. Although it is becoming accepted that Wieser’s classification
may not accurately represent real feeding groups, which are more complex Jensen, 1987a; Moens and Vincx, 1997, it is still considered ecologically informative Thistle et
al., 1995. In order to examine treatment effects on the trophic composition of other nematode species, Diplolaimella sp., a non-selective deposit feeder Type 1B, which
occurred in extremely high numbers in the detritus-enriched samples, is not included in Fig. 7. A general trend was observed that the highest numbers of non-selective deposit
feeders Type 1B shifted from controls C on day 1 to low dose samples L on day 10 then to medium dose samples M on day 30 and, finally, to high dose samples H on
day 60 post-placement. Other groups showed a similar trend but with a time lag. For example, epi-growth feeders Type 2A had the highest numbers in the control from
days 1 to 10 post-placement. After 30 days, numbers in the control and medium dose samples were both high. The highest numbers occurred in the high dose samples 60 days
post-placement. By the end of the experiment, all the feeding groups had obtained their highest absolute numbers in the high dose samples Fig. 7. The relative abundance of
the four trophic groups were, however, not very different from each other between the
114 H
. Zhou J. Exp. Mar. Biol. Ecol. 256 2001 99 –121 Table 8
SIMPER analysis of the nematode community identifying the top three species making the biggest percentage contributions to Bray–Curtis dissimilarities based on single square root transformed abundance data
between treatments at a specific stage of the experiment. C, control; L, low dose; M, medium dose; H, high dose
Samples 10 Days
30 Days 60 Days
compared Species
Species Species
a a
a
C and L Diplolaimella sp.
15.6 Diplolaimella sp.
28.4 Diplolaimella sp.
21.8
a a
Theristus sp. 11.6
Chromaspirina sp. 5.2
Chromaspirina sp. 6.0
a a
Unidentified species 5.6
Haliplectus wheeleri 3.0
Desmodora cazca 4.4
a a
a
C and M Diplolaimella sp.
13.1 Diplolaimella sp.
18.6 Diplolaimella sp.
37.2
a a
a
Theristus sp. 7.6
Theristus sp. 10.0
Megadesmolaimus sp. 3.5
a
Unidentified species 5.6
Chromaspirina sp. 6.8
Diplolaimelloides sp. 2.9
a a
a
C and H Diplolaimella sp.
8.0 Diplolaimella sp.
23.5 Diplolaimella sp.
35.2
a a
Unidentified species 7.1
Diplolaimelloides sp. 5.5
Anoplostoma viviparum 4.4
a a
a
Diplolaimelloides sp. 6.7
Theristus sp. 5.5
Dichromadora sp. 3.3
a a
L and M Diplolaimella sp.
12.6 Diplolaimella sp.
13.7 Diplolaimella sp.
24.3
a
Theristus sp. 9.3
Theristus sp. 10.3
Chromaspirina sp. 5.3
a a
Diploliamelloides sp. 6.6
Chromaspirina sp. 5.8
Megadesmolaimus sp. 4.3
a
L and H Diplolaimella sp.
12.2 Diplolaimella sp.
16.2 Diplolaimella sp.
27.4
a a
Theristus sp. 10.8
Theristus sp. 7.2
Anoplostoma viviparum 4.7
a a
Diplolaimelloides sp. 8.8
Diplolaimelloides sp. 6.1
Chromaspirina sp. 3.9
a
M and H Diplolaimella sp.
12.1 Theristus sp.
11.1 Diplolaimella sp.
23.1
a a
Theristus sp. 7.3
Diplolaimella sp. 8.0
Anoplostoma viviparum 4.9
a a
Diplolaimellodies sp. 6.3
Chromaspirina sp. 5.9
Paracanthochus sp. 4.1
a
Average density of the species is higher in the latter sample.
field control and different levels of treatment if the two numerically dominant species, namely Parastomanema sp. and Diplolaimella sp., were not included Fig. 8A,B.
4. Discussion
